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Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage

  • Aarti Krishnan
    Footnotes
    Affiliations
    Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva CMU, 1 Rue Michel-Servet, 1211 Geneva 4 Switzerland
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  • Joachim Kloehn
    Affiliations
    Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva CMU, 1 Rue Michel-Servet, 1211 Geneva 4 Switzerland
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  • Matteo Lunghi
    Affiliations
    Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva CMU, 1 Rue Michel-Servet, 1211 Geneva 4 Switzerland
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  • Dominique Soldati-Favre
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva CMU, 1 Rue Michel-Servet, 1211 Geneva 4 Switzerland
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  • Author Footnotes
    1 Supported by Grant 51PHP0_157303, an interdisciplinary Ph.D. program of SystemsX.ch awarded by the Swiss National Science Foundation (SNSF). The authors declare that they have no conflicts of interest with the contents of this article.
Open AccessPublished:November 25, 2019DOI:https://doi.org/10.1016/S0021-9258(17)49928-5
      The Apicomplexa phylum comprises diverse parasitic organisms that have evolved from a free-living ancestor. These obligate intracellular parasites exhibit versatile metabolic capabilities reflecting their capacity to survive and grow in different hosts and varying niches. Determined by nutrient availability, they either use their biosynthesis machineries or largely depend on their host for metabolite acquisition. Because vitamins cannot be synthesized by the mammalian host, the enzymes required for their synthesis in apicomplexan parasites represent a large repertoire of potential therapeutic targets. Here, we review recent advances in metabolic reconstruction and functional studies coupled to metabolomics that unravel the interplay between biosynthesis and salvage of vitamins and cofactors in apicomplexans. A particular emphasis is placed on Toxoplasma gondii, during both its acute and latent stages of infection.

      Introduction

      Members of the Apicomplexa encompass a large number of parasites exhibiting a great level of diversity in their life cycles, with morphologically distinct stages in one or more hosts. The phylum includes coccidians, hemosporidians, piroplasms, Cryptosporidia, and gregarines that occupy divergent niches (
      • Plattner F.
      • Soldati-Favre D.
      Hijacking of host cellular functions by the Apicomplexa.
      ). Toxoplasma gondii is the most successful zoonotic parasite of the cyst-forming subclass of coccidians. The proliferative tachyzoites infect and replicate in most cell types and are responsible for an acute infection, whereas the dormant cyst-forming bradyzoites are responsible for chronic infection, predominantly in the brain and striated muscles (
      • Dubey J.P.
      • Lindsay D.S.
      • Speer C.A.
      Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts.
      ,
      • Dubey J.P.
      Long-term persistence of Toxoplasma gondii in tissues of pigs inoculated with T. gondii oocysts and effect of freezing on viability of tissue cysts in pork.
      ). Plasmodium falciparum is the deadliest form of the human malaria parasites that proliferate in erythrocytes and hepatocytes. T. gondii and malaria parasites replicate intracellularly within a parasitophorous vacuole membrane that is permeable to small metabolites (
      • Polonais V.
      • Soldati-Favre D.
      Versatility in the acquisition of energy and carbon sources by the Apicomplexa.
      ,
      • Gold D.A.
      • Kaplan A.D.
      • Lis A.
      • Bett G.C.L.
      • Rosowski E.E.
      • Cirelli K.M.
      • Bougdour A.
      • Sidik S.M.
      • Beck J.R.
      • Lourido S.
      • Egea P.F.
      • Bradley P.J.
      • Hakimi M.-A.
      • Rasmusson R.L.
      • Saeij J.P.J.
      The toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole.
      ,
      • Garten M.
      • Nasamu A.S.
      • Niles J.C.
      • Zimmerberg J.
      • Goldberg D.E.
      • Beck J.R.
      EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX.
      ,
      • Sherling E.S.
      • van Ooij C.
      Host cell remodeling by pathogens: the exomembrane system in Plasmodium-infected erythrocytes.
      ,
      • Baumeister S.
      • Winterberg M.
      • Duranton C.
      • Huber S.M.
      • Lang F.
      • Kirk K.
      • Lingelbach K.
      Evidence for the involvement of Plasmodium falciparum proteins in the formation of new permeability pathways in the erythrocyte membrane.
      ). In contrast, Theileria and Babesia species that belong to the genera of piroplasms rapidly escape the vacuole and proliferate freely in the cytoplasm of lymphocytes and red blood cells, respectively, with a more direct access to host nutrients (
      • Mehlhorn H.
      • Shein E.
      The piroplasms: life cycle and sexual stages.
      ,
      • Jalovecka M.
      • Hajdusek O.
      • Sojka D.
      • Kopacek P.
      • Malandrin L.
      The complexity of piroplasms life cycles.
      ). Cryptosporidium, an enteric pathogen that relies only on a single host for both its sexual and asexual reproduction, develops in an extracytoplasmic compartment confined to the apical surfaces of epithelial cells and in a vacuole connected to the host cell via an extensively folded membrane structure called the feeder organelle (
      • O'Hara S.P.
      • Chen X.-M.
      The cell biology of Cryptosporidium infection.
      ). In humans, the causative agents of malaria, toxoplasmosis, and cryptosporidiosis are responsible for over a million deaths each year. From an evolutionary point of view, it is useful to compare the needs and capabilities between the closely related alveolates from the Apicomplexa and Chromerida phylum that group species capable of photosynthesis (
      • Moore R.B.
      • Oborník M.
      • Janouškovec J.
      • Chrudimský T.
      • Vancová M.
      • Green D.H.
      • Wright S.W.
      • Davies N.W.
      • Bolch C.J.S.
      • Heimann K.
      • Šlapeta
      • Hoegh-Guldberg J.O.
      • Logsdon J.M.
      • Carter D.A.
      A photosynthetic alveolate closely related to apicomplexan parasites.
      ).
      Our knowledge of apicomplexan metabolism has greatly benefited from the assembly of parasite genomes and has advanced through functional studies, in particular of T. gondii and Plasmodium spp. A necessary step toward a global understanding of the central carbon metabolism as well as the synthesis and uptake of amino acids, lipids, vitamins, and cofactors involves the use of in silico methods capable of predicting essential reactions, genes, and synthetic lethal pairs (
      • Song C.
      • Chiasson M.A.
      • Nursimulu N.
      • Hung S.S.
      • Wasmuth J.
      • Grigg M.E.
      • Parkinson J.
      Metabolic reconstruction identifies strain-specific regulation of virulence in Toxoplasma gondii.
      ,
      • Chiappino-Pepe A.
      • Tymoshenko S.
      • Ataman M.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.
      ,
      • Tymoshenko S.
      • Oppenheim R.D.
      • Agren R.
      • Nielsen J.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Metabolic needs and capabilities of Toxoplasma gondii through combined computational and experimental analysis.
      ,
      • Stanway R.R.
      • Bushell E.
      • Chiappino-Pepe A.
      • Roques M.
      • Sanderson T.
      • Franke-Fayard B.
      • Caldelari R.
      • Golomingi M.
      • Nyonda M.
      • Pandey V.
      • Schwach F.
      • Chevalley S.
      • Ramesar J.
      • Metcalf T.
      • Herd C.
      • et al.
      Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.
      ).
      Krishnan, et al., Functional and computational genomics reveal unprecedented flexibility in stage-specific Toxoplasma metabolism. Cell Host & Microbe., in press.
      Currently available genome-scale computational models for T. gondii and the malaria parasites (
      • Chiappino-Pepe A.
      • Tymoshenko S.
      • Ataman M.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.
      ,
      • Tymoshenko S.
      • Oppenheim R.D.
      • Agren R.
      • Nielsen J.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Metabolic needs and capabilities of Toxoplasma gondii through combined computational and experimental analysis.
      ,
      • Stanway R.R.
      • Bushell E.
      • Chiappino-Pepe A.
      • Roques M.
      • Sanderson T.
      • Franke-Fayard B.
      • Caldelari R.
      • Golomingi M.
      • Nyonda M.
      • Pandey V.
      • Schwach F.
      • Chevalley S.
      • Ramesar J.
      • Metcalf T.
      • Herd C.
      • et al.
      Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.
      ,
      • Sidik S.M.
      • Huet D.
      • Ganesan S.M.
      • Huynh M.-H.H.
      • Wang T.
      • Nasamu A.S.
      • Thiru P.
      • Saeij J.P.J.
      • Carruthers V.B.
      • Niles J.C.
      • Lourido S.
      A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.
      )3 have recently been challenged by an impressive series of genome-wide gene fitness screens (
      • Sidik S.M.
      • Huet D.
      • Ganesan S.M.
      • Huynh M.-H.H.
      • Wang T.
      • Nasamu A.S.
      • Thiru P.
      • Saeij J.P.J.
      • Carruthers V.B.
      • Niles J.C.
      • Lourido S.
      A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.
      ,
      • Zhang M.
      • Wang C.
      • Otto T.D.
      • Oberstaller J.
      • Liao X.
      • Adapa S.R.
      • Udenze K.
      • Bronner I.F.
      • Casandra D.
      • Mayho M.
      • Brown J.
      • Li S.
      • Swanson J.
      • Rayner J.C.
      • Jiang R.H.Y.
      • Adams J.H.
      Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.
      ,
      • Bushell E.
      • Gomes A.R.
      • Sanderson T.
      • Anar B.
      • Girling G.
      • Herd C.
      • Metcalf T.
      • Modrzynska K.
      • Schwach F.
      • Martin R.E.
      • Mather M.W.
      • McFadden G.I.
      • Parts L.
      • Rutledge G.G.
      • Vaidya A.B.
      • et al.
      Functional profiling of a Plasmodium genome reveals an abundance of essential genes.
      ) and stage-specific transcriptomics data (
      • Hehl A.B.
      • Basso W.U.
      • Lippuner C.
      • Ramakrishnan C.
      • Okoniewski M.
      • Walker R.A.
      • Grigg M.E.
      • Smith N.C.
      • Deplazes P.
      Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.
      ,
      • Otto T.D.
      • Böhme U.
      • Jackson A.P.
      • Hunt M.
      • Franke-Fayard B.
      • Hoeijmakers W.A.M.
      • Religa A.A.
      • Robertson L.
      • Sanders M.
      • Ogun S.A.
      • Cunningham D.
      • Erhart A.
      • Billker O.
      • Khan S.M.
      • Stunnenberg H.G.
      • et al.
      A comprehensive evaluation of rodent malaria parasite genomes and gene expression.
      ,
      • Caldelari R.
      • Dogga S.
      • Schmid M.W.
      • Franke-Fayard B.
      • Janse C.J.
      • Soldati-Favre D.
      • Heussler V.
      Transcriptome analysis of Plasmodium berghei during exo-erythrocytic development.
      ). These global approaches have turned out to be instrumental for the curation and validation of computational networks. Ultimately, incorporating functional analyses of metabolic pathways with molecular biology and metabolomic techniques will improve the accuracy of computational predictions.
      In the recent past, several studies have illustrated the power of combining genetic and metabolomics approaches to understand metabolic functions in T. gondii. To summarize, it was shown that glucose and glutamine are the major carbon sources utilized by T. gondii tachyzoites (
      • MacRae J.I.I.
      • Sheiner L.
      • Nahid A.
      • Tonkin C.
      • Striepen B.
      • McConville M.J.J.
      Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii.
      ,
      • Nitzsche R.
      • Zagoriy V.
      • Lucius R.
      • Gupta N.
      Metabolic cooperation of glucose and glutamine is essential for the lytic cycle of obligate intracellular parasite Toxoplasma gondii.
      ) and that glycolysis is essential for bradyzoites (
      • Shukla A.
      • Olszewski K.L.
      • Llinás M.
      • Rommereim L.M.
      • Fox B.A.
      • Bzik D.J.
      • Xia D.
      • Wastling J.
      • Beiting D.
      • Roos D.S.
      • Shanmugam D.
      Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii.
      ). The gluconeogenic enzyme fructose bisphosphatase was essential to regulate glycolytic flux in a futile cycle with phosphofructokinase (
      • Blume M.
      • Nitzsche R.
      • Sternberg U.
      • Gerlic M.
      • Masters S.L.
      • Gupta N.
      • McConville M.J.
      A Toxoplasma gondii gluconeogenic enzyme contributes to robust central carbon metabolism and is essential for replication and virulence.
      ). Uniquely, acetyl-CoA in the mitochondrion was shown to be produced via the branched-chain α-ketoacid dehydrogenase complex and not the canonical pyruvate dehydrogenase (PDH)
      The abbreviations used are: PDH
      pyruvate dehydrogenase
      TPK
      thiamine diphosphokinase
      PBAL
      pantoate-β-alanine ligase or PAN synthase
      PAN
      pantothenate
      PLP
      pyridoxal 5′-phosphate
      PLK
      pyridoxal kinase or PdxK
      CPO
      coproporphyrinogen oxidase
      CPDH
      coproporphyrinogen dehydrogenase
      FC
      ferrochelatase
      FA
      fatty acid
      TPP
      thiamine pyrophosphate
      FS
      fitness score(s)
      ETC
      electron transport chain
      KPHMT
      Ketopantoate hydroxymethyltransferase
      KPR
      α-ketopantoate reductase
      PanK
      pantothenate kinase
      DOXP
      1-deoxy-d-xylulose 5-phosphate
      ACCase
      acetyl-CoA carboxylase
      ALA
      δ-aminolevulinic acid
      ProtoIX
      protoporphyrin IX.
      complex (
      • Oppenheim R.D.
      • Creek D.J.
      • Macrae J.I.
      • Modrzynska K.K.
      • Pino P.
      • Limenitakis J.
      • Polonais V.
      • Seeber F.
      • Barrett M.P.
      • Billker O.
      • McConville M.J.
      • Soldati-Favre D.
      BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondiiPlasmodium berghei.
      ). PDH is required for a functional fatty acid (FA) synthase complex, also known as the FASII, in the apicoplast that produces medium-chain FAs, further elongated at the endoplasmic reticulum to form long monounsaturated FAs (
      • Ramakrishnan S.
      • Docampo M.D.
      • Macrae J.I.
      • Pujol F.M.
      • Brooks C.F.
      • van Dooren G.G.
      • Hiltunen J.K.
      • Kastaniotis A.J.
      • McConville M.J.
      • Striepen B.
      • Kalervo J.H.
      • Kastaniotis A.J.
      • McConville M.J.
      • Striepen B.
      Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii.
      ,
      • Ramakrishnan S.
      • Docampo M.D.
      • MacRae J.I.
      • Ralton J.E.
      • Rupasinghe T.
      • McConville M.J.
      • Striepen B.
      The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell.
      ).
      Given the availability of large-scale data sets, systems-wide analysis of parasite metabolism offers a great opportunity to identify essential metabolic functions for targeted drug intervention. In a recent study,3 a well-curated computational genome-scale model, iTgo (in silico T. gondii), was generated. iTgo contains 556 metabolic genes and integrates all available data sets to serve as a valuable platform for model-guided investigations. To harmonize the model with the genome-wide fitness scores for metabolic genes, additional constraints on substrate availabilities from the host as well as reaction utilization based on transcriptomics data were applied (
      • Stanway R.R.
      • Bushell E.
      • Chiappino-Pepe A.
      • Roques M.
      • Sanderson T.
      • Franke-Fayard B.
      • Caldelari R.
      • Golomingi M.
      • Nyonda M.
      • Pandey V.
      • Schwach F.
      • Chevalley S.
      • Ramesar J.
      • Metcalf T.
      • Herd C.
      • et al.
      Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.
      ,
      • Pandey V.
      • Hernandez Gardiol D.
      • Chiappino Pepe A.
      • Hatzimanikatis V.
      TEX-FBA: a constraint-based method for integrating gene expression, thermodynamics, and metabolomics data into genome-scale metabolic models.
      ). The workflow led to a model, 80% consistent with experimentally observed phenotypes,3 allowing for reliable hypothesis generation for experimental validation. The two previous metabolic reconstructions (
      • Song C.
      • Chiasson M.A.
      • Nursimulu N.
      • Hung S.S.
      • Wasmuth J.
      • Grigg M.E.
      • Parkinson J.
      Metabolic reconstruction identifies strain-specific regulation of virulence in Toxoplasma gondii.
      ,
      • Tymoshenko S.
      • Oppenheim R.D.
      • Agren R.
      • Nielsen J.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Metabolic needs and capabilities of Toxoplasma gondii through combined computational and experimental analysis.
      ) identified several essential metabolic functions and differences within the clonal strains of T. gondii that display distinct virulence profiles. Within the apicomplexans, the most studied and comprehensive metabolic reconstructions were generated for P. falciparum and the rodent malaria parasite, Plasmodium berghei (
      • Chiappino-Pepe A.
      • Tymoshenko S.
      • Ataman M.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.
      ,
      • Stanway R.R.
      • Bushell E.
      • Chiappino-Pepe A.
      • Roques M.
      • Sanderson T.
      • Franke-Fayard B.
      • Caldelari R.
      • Golomingi M.
      • Nyonda M.
      • Pandey V.
      • Schwach F.
      • Chevalley S.
      • Ramesar J.
      • Metcalf T.
      • Herd C.
      • et al.
      Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.
      ,
      • Tymoshenko S.
      • Oppenheim R.D.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Functional genomics of Plasmodium falciparum using metabolic modelling and analysis.
      ). Constant modeling efforts with the incorporation of physiological parameters, such as metabolomics and fluxomics, continue to expand our knowledge of the metabolic versatility of the apicomplexans. Although challenging, future models should consider the kinetic properties of reactions, allowing the simulation of altered enzymatic activities in both the host and parasite (
      • Tymoshenko S.
      • Oppenheim R.D.
      • Soldati-Favre D.
      • Hatzimanikatis V.
      Functional genomics of Plasmodium falciparum using metabolic modelling and analysis.
      ). Ideally, as complementary constituents of an iterative process, both computational and experimental efforts will ultimately lead to the identification of potential drug targets, mechanisms of drug action and complex host-pathogen interactions.
      Among the indispensable pathways for parasite proliferation and persistence, the biosynthesis of vitamins and cofactors offers potential targets for intervention. Vitamins are essential precursors for the production of cofactors and, in humans, can be acquired solely through the diet (
      • Bender D.A.
      ). To date, 13 metabolites are classified as vitamins, required for the functioning of a mammalian cell, facilitating numerous enzymatic reactions. Nine of the 13 vitamins are known to be utilized by the apicomplexans, with three of them (vitamins B5, B6, and B9) being de novo–synthesized by some parasites (
      • Müller S.
      • Kappes B.
      Vitamin and cofactor biosynthesis pathways in Plasmodium and other apicomplexan parasites.
      ). The vitamins that can be synthesized de novo are probably low in abundance in one or more niches and cannot be sufficiently salvaged. Comparison across the phylum can reveal interesting insights into the origins and subsequent loss of several pathways in certain genera, such as the Cryptosporidia and piroplasms (
      • Seeber F.
      • Soldati-Favre D.
      Metabolic pathways in the apicoplast of Apicomplexa.
      ,
      • Hung S.S.
      • Parkinson J.
      Post-genomics resources and tools for studying apicomplexan metabolism.
      ,
      • Fleige T.
      • Limenitakis J.
      • Soldati-Favre D.
      Apicoplast: keep it or leave it.
      ) (Fig. 1). Both genera possess limited biosynthesis capabilities, reflecting their lifestyle in a nutrient-rich environment and adaptation to mechanisms for metabolite acquisition from the host. Concordantly, the genome of Cryptosporidium hominis was shown to encode more than 80 genes with strong similarity to known transporters and several hundred genes with transporter-like properties (
      • Xu P.
      • Widmer G.
      • Wang Y.
      • Ozaki L.S.
      • Alves J.M.
      • Serrano M.G.
      • Puiu D.
      • Manque P.
      • Akiyoshi D.
      • Mackey A.J.
      • Pearson W.R.
      • Dear P.H.
      • Bankier A.T.
      • Peterson D.L.
      • Abrahamsen M.S.
      • Kapur V.
      • Tzipori S.
      • Buck G.A.
      The genome of Cryptosporidium hominis.
      ). Cryptosporidia are also in close contact with the microbiome in the intestinal gut, thus expanding their capacity for nutrient acquisition (
      • Burgess S.L.
      • Gilchrist C.A.
      • Lynn T.C.
      • Petri W.A.
      Parasitic protozoa and interactions with the host intestinal microbiota.
      ).
      Figure thumbnail gr1
      Figure 1Conservation of vitamin and cofactor biosynthesis or scavenge pathways within the apicomplexans and the human host. The presence or absence of metabolic pathways within the Apicomplexan and Chromerida phylum and the human host, Homo sapiens, is summarized. The gene identifiers and enzyme names in each pathway can be found in . For each genus, representative organisms were chosen: coccidians (T. gondii), hemosporidians (P. falciparum), piroplasms (Babesia bovis and Theileria annulata), Cryptosporidia (Cryptosporidium muris), gregarines (Gregarina niphandrodes), and chromerida (C. velia and V. brassicaformis).
      In the next sections, we review the progress made in T. gondii and apicomplexans in general, to better understand the interrelationship of de novo synthesis and scavenge routes for vitamins and cofactors and their utilization in different life cycle stages. An overview of the pathways in both T. gondii and its mammalian host is presented in Fig. 2. Further, the latest observations are discussed in the context of long-standing questions on the roles of the metabolic pathways for latency and their potential as drug targets.
      Figure thumbnail gr2
      Figure 2Vitamins and cofactors biosynthesis versus scavenge pathways in T. gondii and its mammalian host. Metabolites that can either be de novo produced (blue) or must be salvaged (pink) from an external source are depicted. Enzymes for the production of metabolites (boldface blue type) are potential drug targets, given the unique synthesis capability of the parasite, but not the host.

      Vitamin B1

      Vitamin B1, or thiamine, is an important precursor for its metabolically active form, thiamine pyrophosphate (TPP). TPP acts as a cofactor for enzymes implicated in carbohydrate and amino acid metabolism, such as the PDH complex, 2-oxoglutarate dehydrogenase, pyruvate decarboxylase, and dihydrolipoamide dehydrogenase. In T. gondii, these enzymes are either residents of the secondary endosymbiotic organelle, called the apicoplast, or the mitochondrion, suggesting a need for the cofactor within these subcellular compartments. Like their mammalian host, the parasites do not possess the pathway for thiamine biosynthesis and must therefore acquire it. Hemosporidians (in particular P. falciparum) are the only apicomplexans that possess the enzymes to synthesize thiamine, like bacteria, plants, and fungi (
      • Hellgren O.
      • Bensch S.
      • Videvall E.
      De novo synthesis of thiamine (vitamin B1) is the ancestral state in Plasmodium parasites—evidence from avian haemosporidians.
      ,
      • Wrenger C.
      • Eschbach M.-L.
      • Müller I.B.
      • Laun N.P.
      • Begley T.P.
      • Walter R.D.
      Vitamin B1de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine.
      ,
      • Wrenger C.
      • Knöckel J.
      • Walter R.D.
      • Müller I.B.
      Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?.
      ). The genes implicated in the synthesis of TPP are, however, expressed only in the mosquito vector (salivary gland sporozoites) stage (
      • Tarun A.S.
      • Baer K.
      • Dumpit R.F.
      • Gray S.
      • Lejarcegui N.
      • Frevert U.
      • Kappe S.H.I.
      Quantitative isolation and in vivo imaging of malaria parasite liver stages.
      ). Despite the ability to synthesize thiamine, Plasmodium spp., like other apicomplexans, harbor the key enzyme thiamine diphosphokinase (TPK) to convert the scavenged thiamine into TPP. TPK is expressed in all stages of the Plasmodium life cycle, and several studies have shown that parasite replication is inhibited by thiamine analogues that generate toxic anti-metabolites (
      • Chan X.W.A.
      • Wrenger C.
      • Stahl K.
      • Bergmann B.
      • Winterberg M.
      • Müller I.B.
      • Saliba K.J.
      • Muller I.B.
      • Saliba K.J.
      Chemical and genetic validation of thiamine utilization as an antimalarial drug target.
      ,
      • Zilles J.L.
      • Croal L.R.
      • Downs D.M.
      Action of the thiamine antagonist bacimethrin on thiamine biosynthesis.
      ). Deduced from the genome-wide CRISPR-Cas9 screen for T. gondii performed in vitro, TPK is critical for in vitro tachyzoite survival with a high negative fitness score (FS) (−3.28) (Fig. 3). FS are experimentally observed measures (ranging from −7 to +3) and assess the fitness cost of a given gene for parasite survival (
      • Sidik S.M.
      • Huet D.
      • Ganesan S.M.
      • Huynh M.-H.H.
      • Wang T.
      • Nasamu A.S.
      • Thiru P.
      • Saeij J.P.J.
      • Carruthers V.B.
      • Niles J.C.
      • Lourido S.
      A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.
      ).
      Figure thumbnail gr3
      Figure 3The scavenge pathways and bioconversion of vitamins (B1, B2, B3, and B7). T. gondii must uptake vitamins B1, B2, B3, and B7 via unknown transport mechanisms and subsequently convert them into the cofactors for utilization within the parasite. FS for the enzymes for the bioconversion are color-coded (in circles). NPPRT, nicotinate phosphoribosyltransferase; NNAT, nicotinate-nucleotide adenylyl transferase.
      The mechanism by which thiamine is taken up and translocated across organelles where it is needed is yet to be determined. In humans the thiamine transporters, hThTr1 and hThTr2 have been well-characterized (
      • Rajgopal A.
      • Edmondnson A.
      • Goldman I.D.
      • Zhao R.
      SLC19A3 encodes a second thiamine transporter ThTr2.
      ,
      • Subramanian V.S.
      • Marchant J.S.
      • Parker I.
      • Said H.M.
      Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms.
      ), but no obvious orthologs within the parasite's genome could be identified. Interestingly, in certain apicomplexans, such as Cryptosporidia and piroplasms, salvage of the phosphorylated form (TPP) must occur.

      Vitamin B2

      Vitamin B2, or riboflavin, is crucial for flavin-dependent processes occurring in all subcellular compartments. FMN and FAD participate in redox reactions and play an essential role for the proper functioning of the electron transport chain (ETC), tricarboxylic acid cycle, and fatty acid biosynthesis. Like their mammalian hosts, most apicomplexans are unable to synthesize riboflavin but possess the capacity to convert riboflavin into FMN and FAD. The two genes coding for their synthesis, riboflavin kinase and FAD synthase, are present in T. gondii and are fitness-conferring with an FS of −3.97 and −4.87, respectively (Fig. 3). Exceptionally, Cryptosporidia appear to lack these enzymes and therefore must take up both FMN and FAD, suggesting an exquisite adaptability to scavenge phosphorylated cofactors. Outside the Apicomplexa phylum, the absence of an FMN/FAD synthase can be seen in obligate intracellular α-proteobacteria, Rickettsiae (
      • Driscoll T.P.
      • Verhoeve V.I.
      • Guillotte M.L.
      • Lehman S.S.
      • Rennoll S.A.
      • Beier-Sexton M.
      • Rahman M.S.
      • Azad A.F.
      • Gillespie J.J.
      Wholly Rickettsia! Reconstructed metabolic profile of the quintessential bacterial parasite of eukaryotic cells.
      ).

      Vitamin B3

      Vitamin B3, or nicotinic acid, also known as niacin, is essential for generation of coenzymes NAD+ and NADP+, which act as key electron carriers in a cell. The apicomplexans are unable to synthesize nicotinate or nicotinamide de novo, indicating that these metabolites are salvaged from the host. All apicomplexans possess the enzymes for the subsequent conversion into NAD+ and NADP+, although the corresponding genes appear dispensable for T. gondii tachyzoites, based on their FS (NAD+ synthase, +0.03; NAD+ kinase, −1.33) (Fig. 3). Of relevance, the CRISPR-Cas9 screen was performed with cultured human foreskin fibroblasts grown in rich media containing an abundance of amino acids, vitamins, salts, and sugars. This may allow certain genes to seem dispensable than they actually would be in a physiological environment more restricted in nutrients. Fitness scores might also vary, depending on the metabolic rates and capabilities of different host cell types (in vitro or in vivo).
      In P. falciparum, infected erythrocytes showed a 10-fold increase in NAD+ content compared with uninfected cells, suggesting an efficient and functional biosynthesis pathway in the parasite (
      • Zerez C.R.
      • Roth Jr., E.F.
      • Schulman S.
      • Tanaka K.R.
      Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes.
      ). Due to the substantial release of NAD+ from Plasmodium-infected erythrocytes, NAD+ has been proposed as a potential clinical biomarker for malaria (
      • Beri D.
      • Ramdani G.
      • Balan B.
      • Gadara D.
      • Poojary M.
      • Momeux L.
      • Tatu U.
      • Langsley G.
      Insights into physiological roles of unique metabolites released from Plasmodium-infected RBCs and their potential as clinical biomarkers for malaria.
      ). The impact of blocking the parasite nicotinate mononucleotide adenylyl transferase, which synthesizes NAD+ from nicotinate, validates the biosynthesis pathway as an antimalarial target (
      • O'Hara J.K.
      • Kerwin L.J.
      • Cobbold S.A.
      • Tai J.
      • Bedell T.A.
      • Reider P.J.
      • Llinás M.
      Targeting NAD+ metabolism in the human malaria parasite Plasmodium falciparum.
      ).

      Vitamin B5

      Vitamin B5, or pantothenate (PAN), is the precursor for the biosynthesis of the essential cofactor, CoA. PAN synthesis takes place in most bacteria plants and fungi, but not in animals. The biosynthesis of CoA from PAN, on the other hand, is present in almost all organisms. The de novo synthesis of PAN requires three enzymatic activities: hydroxymethyl transfer to ketoisovalerate (KPHMT), α-ketopantoate reduction (KPR) to pantoate, and pantoate-β-alanine ligation (PBAL). Interestingly, T. gondii encodes the pathway in two sequences conserved within the coccidians, which include Hammondia, Neospora, Besnoitia, Cyclospora, and Eimeria genera. The PAN synthesis pathway has been partially characterized in T. gondii, and its essentiality has been proposed based on the use of chemical inhibitors (
      • Mageed S.N.
      • Cunningham F.
      • Hung A.W.
      • Silvestre H.L.
      • Wen S.
      • Blundell T.L.
      • Abell C.
      • McConkey G.A.
      Pantothenic acid biosynthesis in the parasite Toxoplasma gondii: a target for chemotherapy.
      ). However, the tested drugs had been developed for Mycobacterium tuberculosis homologue (panC), and off-target effects cannot be excluded.
      In T. gondii, the first enzyme in the PAN synthesis pathway is bifunctional, encoding the first two enzymatic steps ketopantoate hydroxymethyl transferase and ketopantoate reductase (KPHMT-KPR). The KPHMT and KPR domains of the protein present conserved key catalytic residues (
      • Ciulli A.
      • Chirgadze D.Y.
      • Smith A.G.
      • Blundell T.L.
      • Abell C.
      Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound.
      ,
      • von Delft F.
      • Inoue T.
      • Saldanha S.A.
      • Ottenhof H.H.
      • Schmitzberger F.
      • Birch L.M.
      • Dhanaraj V.
      • Witty M.
      • Smith A.G.
      • Blundell T.L.
      • Abell C.
      Structure of E. coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites.
      ) when compared with Escherichia coli panB and panE, respectively. The fusion of the two catalytic domains into one ORF can also be found outside the Apicomplexa phylum in Dinoflagellates (Perkinsus marinus) and free-living photosynthetic Chromerida (Vitrella brassicaformis and Chromera velia), where, interestingly, a single ORF comprises all three enzymes for the synthesis of PAN (Table S1).
      The final step of PAN synthesis is catalyzed by PBAL, which ligates pantoate with β-alanine. Sequence comparison with E. coli panC indicates that >30% of the catalytic domain and all catalytic residues (
      • von Delft F.
      • Lewendon A.
      • Dhanaraj V.
      • Blundell T.L.
      • Abell C.
      • Smith A.G.
      The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily.
      ) are conserved in T. gondii PBAL, pointing to a possible conservation of function. In all members of the phylum, the protein presents an extended N and C terminus (the latter conserved >45% within Neospora, Hammondia, and Besnoitia genera), although no known molecular function has been associated to date. The respective FS of KPHMT-KPR (+0.09) and PBAL (+0.72) indicate in vitro dispensability for PAN synthesis, suggesting that T. gondii, as demonstrated for P. falciparum (
      • Saliba K.J.
      • Horner H.A.
      • Kirk K.
      Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum.
      ), utilizes host derived PAN for CoA synthesis. Except for the coccidians, the apicomplexans lack PAN synthesis enzymes, and attempts to identify a PAN transporter by orthology have proven difficult (
      • Augagneur Y.
      • Jaubert L.
      • Schiavoni M.
      • Pachikara N.
      • Garg A.
      • Usmani-Brown S.
      • Wesolowski D.
      • Zeller S.
      • Ghosal A.
      • Cornillot E.
      • Said H.M.
      • Kumar P.
      • Altman S.
      • Ben Mamoun C.
      Identification and functional analysis of the primary pantothenate transporter, PfPAT, of the human malaria parasite Plasmodium falciparum.
      ,
      • Kehrer J.
      • Singer M.
      • Lemgruber L.
      • Silva P.A.G.C.
      • Frischknecht F.
      • Mair G.R.
      A putative small solute transporter is responsible for the secretion of G377 and TRAP-containing secretory vesicles during Plasmodium gamete egress and sporozoite motility.
      ,
      • Hammoudi P.-M.
      • Maco B.
      • Dogga S.K.
      • Frénal K.
      • Soldati-Favre D.
      Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis.
      ).
      CoA, the end product of the pathway, is essential for a broad range of metabolic functions. It provides activated acyl groups for various metabolic pathways, such as the tricarboxylic acid cycle, fatty acid synthesis, and heme synthesis, as well as for gene regulation and post-translational modification of proteins (
      • Pietrocola F.
      • Galluzzi L.
      • Bravo-San Pedro J.M.
      • Madeo F.
      • Kroemer G.
      Acetyl coenzyme A: a central metabolite and second messenger.
      ). Pantothenate kinase (PanK), which catalyzes the first step in CoA synthesis, has been extensively characterized in P. falciparum (
      • Tjhin E.T.
      • Spry C.
      • Sewell A.L.
      • Hoegl A.
      • Barnard L.
      • Sexton A.E.
      • Siddiqui G.
      • Howieson V.M.
      • Maier A.G.
      • Creek D.J.
      • Strauss E.
      • Marquez R.
      • Auclair K.
      • Saliba K.J.
      Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues.
      ), allowing pantothenamides (pantothenate mimetic compounds) to be catabolized into CoA antimetabolites (
      • de Villiers M.
      • Spry C.
      • Macuamule C.J.
      • Barnard L.
      • Wells G.
      • Saliba K.J.
      • Strauss E.
      Antiplasmodial mode of action of pantothenamides: pantothenate kinase serves as a metabolic activator not as a target.
      ) with deleterious effects for the parasite (
      • Schalkwijk J.
      • Allman E.L.
      • Jansen P.A.
      • Vries L.E
      • de Jackowski S.
      • Botman P.N.
      • Beuckens-Schortinghuis C.A.
      • Koolen K.M.
      • Bolscher J.M.
      • Vos M.W.
      • Miller K.
      • Reeves S.
      • Pett H.
      • Trevitt G.
      • Wittlin S.
      • et al.
      Antimalarial pantothenamide metabolites target acetyl-CoA synthesis in Plasmodium falciparum.
      ). Interestingly, of the five enzymes required for CoA synthesis, phosphopantetheine-cysteine ligase and phosphopantothenoylcysteine decarboxylase, which catalyze the second and third step, respectively, are dispensable in both the rodent malaria parasites Plasmodium yoelii and P. berghei (
      • Bushell E.
      • Gomes A.R.
      • Sanderson T.
      • Anar B.
      • Girling G.
      • Herd C.
      • Metcalf T.
      • Modrzynska K.
      • Schwach F.
      • Martin R.E.
      • Mather M.W.
      • McFadden G.I.
      • Parts L.
      • Rutledge G.G.
      • Vaidya A.B.
      • et al.
      Functional profiling of a Plasmodium genome reveals an abundance of essential genes.
      ,
      • Hart R.J.
      • Abraham A.
      • Aly A.S.I.
      Genetic characterization of coenzyme A biosynthesis reveals essential distinctive functions during malaria parasite development in blood and mosquito.
      ). This observation could be explained by the promiscuous activity of PanK (
      • de Villiers M.
      • Barnard L.
      • Koekemoer L.
      • Snoep J.L.
      • Strauss E.
      Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis.
      ), allowing usage of pantetheine (an intermediate) scavenged from the host cell (
      • Srinivasan B.
      • Baratashvili M.
      • van der Zwaag M.
      • Kanon B.
      • Colombelli C.
      • Lambrechts R.A.
      • Schaap O.
      • Nollen E.A.
      • Podgoršek A.
      • Kosec G.
      • Petković H.
      • Hayflick S.
      • Tiranti V.
      • Reijngoud D.-J.
      • Grzeschik N.A.
      • Sibon O.C.M.
      Extracellular 4′-phosphopantetheine is a source for intracellular coenzyme A synthesis.
      ). In T. gondii, the FS of all of the enzymes of the CoA synthesis pathway indicate essentiality (including the two different genes encoding for PanK) (Fig. 4). We have recently identified the gene coding for the final step, dephospho-CoA kinase, previously thought to be missing from the genome, and have shown that the activity is essential for parasite survival by conditional disruption.
      M. Lunghi, J. Kloehn, and D. Soldati-Favre, unpublished observations.
      Figure thumbnail gr4
      Figure 4PAN (vitamin B5) and CoA biosynthesis pathway. T. gondii can de novo–synthesize or uptake PAN and subsequently convert it into CoA within the parasite. The bifunctional enzyme for PAN synthesis is shown in blue. FS for the enzymes are color-coded (in circles). BCAT, branched-chain amino acid transaminase; HMT, hydroxymethyltransferase; PPCL, phosphopantetheine-cysteine ligase; PPCD, phosphopantothenoylcysteine decarboxylase; PPAT, pantetheine-phosphate adenylyl transferase.
      Taken together, it appears that most apicomplexans share the capability to scavenge PAN from their host. Hence, the retention of the PAN synthesis pathway among the coccidians is intriguing. It is likely that PAN synthesis is required in life cycle stages where exogeneous PAN levels are limiting, such as in sporozoites or in the cyst-enclosed bradyzoites of T. gondii. Importantly, PAN synthesis requires β-alanine, for which no synthesis pathway has been clearly identified in the genome of T. gondii. Thus, the parasite would have to acquire this metabolite from its environment. Further research is necessary to delineate the relevance of PAN synthesis in coccidians.

      Vitamin B6

      Vitamin B6 is part of the essential vitamin B group of molecules, consisting of pyridoxal, pyridoxamine, and pyridoxine. The metabolically active form is pyridoxal 5′-phosphate (PLP). PLP is a crucial cofactor for the activity of over 140 enzymes, several of them involved in amino acid metabolism (
      • Percudani R.
      • Peracchi A.
      A genomic overview of pyridoxal-phosphate-dependent enzymes.
      ,
      • Hoegl A.
      • Nodwell M.B.
      • Kirsch V.C.
      • Bach N.C.
      • Pfanzelt M.
      • Stahl M.
      • Schneider S.
      • Sieber S.A.
      Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.
      ). Two different routes for the de novo synthesis of PLP exist in organisms: 1-deoxy-d-xylulose 5-phosphate (DOXP)-dependent and DOXP-independent (
      • Fitzpatrick T.B.
      • Amrhein N.
      • Kappes B.
      • Macheroux P.
      • Tews I.
      • Raschle T.
      Two independent routes of de novo vitamin B6 biosynthesis: not that different after all.
      ). The DOXP-dependent route occurs in proteobacteria and most other bacteria, whereas eukaryotes, including the apicomplexans, utilize the DOXP-independent route. In this route, PLP is synthesized via the activity of two enzymes, PDX1 (PLP synthase subunit) and PDX2 (class I glutamine amidotransferase). Free vitamin B6 forms can also be phosphorylated via the action of pyridoxal kinase (PLK or PDXK). The subsequent conversion of pyridoxamine-5P and pyridoxine-5P to PLP can be performed via a different enzyme, pyridoxal 5′-phosphate synthase (PLP synthase).
      Both coccidians and hemosporidians possess all of the enzymes for de novo synthesis as well as scavenge of the vitamin (
      • Knöckel J.
      • Müller I.B.
      • Bergmann B.
      • Walter R.D.
      • Wrenger C.
      The apicomplexan parasite Toxoplasma gondii generates pyridoxal phosphate de novo.
      ,
      • Gengenbacher M.
      • Fitzpatrick T.B.
      • Raschle T.
      • Flicker K.
      • Sinning I.
      • Müller S.
      • Macheroux P.
      • Tews I.
      • Kappes B.
      Vitamin B6 biosynthesis by the malaria parasite Plasmodium falciparum: biochemical and structural insights.
      ). The FS for the genes coding for PDX1 (+0.59), PDX2 (+0.08), PLP synthase (−0.33), and PLK (−0.41) indicate dispensability in vitro (Fig. 5), indicating redundancy between synthesis and salvage for PLP production. In a recent study, disrupting de novo biosynthesis of PLP via conditional knockdown of PDX1 was detrimental in parasites lacking the PLK gene.3 The synthetic lethality showed that blocking both routes for cofactor generation is deleterious, and several PLP-dependent enzymes must become inactive. One such enzyme is glycogen phosphorylase, which breaks down the storage polysaccharide amylopectin (
      • Palm D.
      • Klein H.W.
      • Schinzel R.
      • Buehner M.
      • Helmreich E.J.M.
      The role of pyridoxal 5′-phosphate in glycogen phosphorylase catalysis.
      ). In T. gondii, loss of glycogen phosphorylase is associated with amylopectin accumulation and lethal for both tachyzoites and bradyzoites (
      • Sugi T.
      • Tu V.
      • Ma Y.
      • Tomita T.
      • Weiss L.M.
      Toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of brain cysts.
      ). Indeed, amylopectin accumulation was observed in mutants depleted of PLP.3 Although PLP requirement for several enzymes is fulfilled with either the biosynthesis or scavenge pathway in vitro, contrastingly, the deletion of PDX1 alone was sufficient to abolish T. gondii virulence in mice.3 This points to limited or insufficient amounts of pyridoxal in the organs or tissues infected with T. gondii in vivo (
      • Van de Kamp J.L.
      • Westrick J.A.
      • Smolen A.
      B6 vitamer concentrations in mouse plasma, erythrocytes and tissues.
      ). The sole reliance on the de novo pathway for PLP production in vivo makes PDX1 an attractive drug target or candidate for an attenuated live vaccine.
      Figure thumbnail gr5
      Figure 5Pyridoxal-5P (vitamin B6) biosynthesis and scavenge pathways. T. gondii can de novo synthesize PLP or uptake the vitamers to subsequently convert them into PLP within the parasite. PLK (in blue) can phosphorylate any of the vitamers—pyridoxal, pyridoxamine, or pyridoxine—and is synthetically lethal with the synthesis enzyme, PDX1. FS for the enzymes for the bioconversion are color-coded (in circles). Experimentally validated enzymes are circled in black.3
      If the biosynthesis pathway is the major route for PLP production in T. gondii in vivo, the presence and role of PLK is puzzling. To test its role during latency, mice infected with parasites lacking PLK were examined for cyst formation.3 No reduction in cyst number was observed, compared with the WT, suggesting its dispensability for the chronic stage. It is plausible that the enzyme has a role during the sexual or oocyst stages, recycling any free pyridoxal in the cell and preventing toxic accumulation of the vitamers. How the vitamers enter the parasite remains unknown, and the absence of the biosynthesis of PLP in Cryptosporidia and piroplasms further indicates an unusual salvage mechanism for the phosphorylated cofactor.
      In P. falciparum, both PLP biosynthesis and salvage pathways have been shown to be functional. The two genes (encoding for PDX1 and PDX2) are expressed throughout the intraerythrocytic and gametocyte development and have been explored as potential drug targets (
      • Cassera M.B.
      • Gozzo F.C.
      • D'Alexandri F.L.
      • Merino E.F.
      • del Portillo H.A.
      • Peres V.J.
      • Almeida I.C.
      • Eberlin M.N.
      • Wunderlich G.
      • Wiesner J.
      • Jomaa H.
      • Kimura E.A.
      • Katzin A.M.
      The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.
      ,
      • Wrenger C.
      • Eschbach M.-L.
      • Müller I.B.
      • Warnecke D.
      • Walter R.D.
      Analysis of the vitamin B6 biosynthesis pathway in the human malaria parasite Plasmodium falciparum.
      ,
      • Müller I.B.
      • Hyde J.E.
      • Wrenger C.
      Vitamin B metabolism in Plasmodium falciparum as a source of drug targets.
      ,
      • Knöckel J.
      • Müller I.B.
      • Butzloff S.
      • Bergmann B.
      • Walter R.D.
      • Wrenger C.
      The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference.
      ). Prodrugs such as pyridoxyl-tryptophan chimeras that interfere with PLP-dependent enzymes and poison the parasite have also been investigated as antimalarials (
      • Kronenberger T.
      • Lindner J.
      • Meissner K.A.
      • Zimbres F.M.
      • Coronado M.A.
      • Sauer F.M.
      • Schettert I.
      • Wrenger C.
      Vitamin B6-dependent enzymes in the human malaria parasite Plasmodium falciparum: a druggable target?.
      ,
      • Müller I.B.
      • Wu F.
      • Bergmann B.
      • Knöckel J.
      • Walter R.D.
      • Gehring H.
      • Wrenger C.
      Poisoning pyridoxal 5-phosphate-dependent enzymes: a new strategy to target the malaria parasite Plasmodium falciparum.
      ). For organisms that lack biosynthesis capabilities, identification of the transporter of pyridoxal and its derivatives would be of significant interest.

      Vitamin B7

      Vitamin B7 or biotin can be synthesized by bacteria, plants, and some fungi, but not by animals. The apicomplexans also lack the biosynthesis capability for biotin. Biotin is an important cofactor for the enzyme acetyl-CoA carboxylase (ACCase), of which ACCase1 was found in the apicoplast of T. gondii (
      • Jelenska J.
      • Crawford M.J.
      • Harb O.S.
      • Zuther E.
      • Haselkorn R.
      • Roos D.S.
      • Gornicki P.
      Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii.
      ). In bacteria, biotin covalently attaches to the ϵ-amino group of specific lysine residues in the carboxylases via the action of a biotin-ligase (
      • Chapman-Smith A.
      • Cronan Jr., J.E.
      Molecular biology of biotin attachment to proteins.
      ). A putative biotin-ACC-ligase, with similarity to the E. coli biotin operon repressor (BirA) was found in the genome of most apicomplexans. If its role is similar to that of BirA for sensing biotin levels and regulating transcription is unknown (
      • Beckett D.
      The Escherichia coli biotin regulatory system: a transcriptional switch.
      ). How biotin is acquired from the host and transported into the apicoplast, where ACCase1 resides, also remains to be understood. Biotin uptake is mediated by solute transporters in prokaryotes (
      • Hebbeln P.
      • Rodionov D.A.
      • Alfandega A.
      • Eitinger T.
      Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module.
      ) and via a monocarboxylate transporter (MCT1) in mammalian cells (
      • Daberkow R.L.
      • White B.R.
      • Cederberg R.A.
      • Griffin J.B.
      • Zempleni J.
      Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells.
      ).

      Vitamin B9

      Vitamin B9 or folate is crucial for DNA replication, cell division, and synthesis of several amino acids. The folate derivative, 5,10-methylenetetrahydrofolate, is essential for the production of dTMP and dUMP nucleotides. In addition to the de novo folate biosynthesis pathway from shikimate and chorismate, most apicomplexans can also salvage folate from the host via dedicated BT1 or FT transporters (
      • Massimine K.M.
      • Doan L.T.
      • Atreya C.A.
      • Stedman T.T.
      • Anderson K.S.
      • Joiner K.A.
      • Coppens I.
      Toxoplasma gondii is capable of exogenous folate transport: a likely expansion of the BT1 family of transmembrane proteins.
      ,
      • Salcedo-Sora J.E.
      • Ochong E.
      • Beveridge S.
      • Johnson D.
      • Nzila A.
      • Biagini G.A.
      • Stocks P.A.
      • O'Neill P.M.
      • Krishna S.
      • Bray P.G.
      • Ward S.A.
      The molecular basis of folate salvage in Plasmodium falciparum: characterization of two folate transporters.
      ) (Fig. 6). The high-affinity folate transporters were shown to take up radiolabeled exogenous folic acid in T. gondii (
      • Massimine K.M.
      • Doan L.T.
      • Atreya C.A.
      • Stedman T.T.
      • Anderson K.S.
      • Joiner K.A.
      • Coppens I.
      Toxoplasma gondii is capable of exogenous folate transport: a likely expansion of the BT1 family of transmembrane proteins.
      ). If folates are taken up to sustain the acute stage of T. gondii, the existence of the biosynthesis pathway is likely relevant for downstream metabolite production or for a different life cycle stage where the parasite encounters limited access to folates or its precursors. Numerous studies have shown the effects of targeting the folate pathway (
      • Hyde J.E.
      Exploring the folate pathway in Plasmodium falciparum.
      ,
      • Nzila A.
      • Ward S.A.
      • Marsh K.
      • Sims P.F.G.
      • Hyde J.E.
      Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy.
      ). Several anti-parasitic drugs are currently in use, such as sulfonamides targeting dihydropteroate synthase in combination with inhibitors of the dihydrofolate reductase-thymidylate synthase. Although the anti-folates are thought to be safe, recent studies in P. falciparum have shown emerging resistance to the once potent drug combination. Future studies would have to unravel the molecular mechanisms of resistance and enable future development of alternative strategies targeting the crucial biosynthesis and scavenge pathways (
      • Heinberg A.
      • Kirkman L.
      The molecular basis of antifolate resistance in Plasmodium falciparum: looking beyond point mutations.
      ). In recent in vivo experiments, the contributions of para-amino benzoic acid (pABA), a precursor for folate synthesis, were also re-examined (
      • Matz J.M.
      • Watanabe M.
      • Falade M.
      • Tohge T.
      • Hoefgen R.
      • Matuschewski K.
      Plasmodium para-aminobenzoate synthesis and salvage resolve avoidance of folate competition and adaptation to host diet.
      ,
      • Mather M.W.
      • Ke H.
      para-Aminobenzoate synthesis versus salvage in malaria parasites.
      ). pABA is synthesized with the action of two enzymes, aminodeoxychorismate synthase and aminodeoxychorismate lyase. The two genes were knocked out in the rodent malaria parasite P. berghei, and the deletions were shown to be dispensable for parasite propagation in mice fed with a conventional diet. However, in mice fed with milk (lacking pABA), the mutants displayed a severe growth phenotype, abolished with the supplementation of pABA (
      • Matz J.M.
      • Watanabe M.
      • Falade M.
      • Tohge T.
      • Hoefgen R.
      • Matuschewski K.
      Plasmodium para-aminobenzoate synthesis and salvage resolve avoidance of folate competition and adaptation to host diet.
      ,
      • Mather M.W.
      • Ke H.
      para-Aminobenzoate synthesis versus salvage in malaria parasites.
      ). In the liver stage, the lack of aminodeoxychorismate synthase was dispensable, suggesting an active salvage, given the folate-rich environment of the liver. The results therefore indicate a combination of salvage and synthesis in Plasmodium parasites, to ensure the folate requirements for the fast-growing asexual stages are met.
      Figure thumbnail gr6
      Figure 6Folate (vitamin B9) and biopterin biosynthesis and scavenge pathways. T. gondii can de novo–synthesize or uptake folates and biopterins. FS for the enzymes for the bioconversion are color-coded (in circles). Enzymes in blue are bifunctional, capable of catalyzing two subsequent reaction steps. TS, thymidylate synthase; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthase; GTP-CH, GTP cyclohydrolase; MDTS1, molybdopterin cofactor synthesis protein 1 (MOCS1); MDTS2, molybdopterin cofactor synthesis protein 2 (MOCS2/MoaE); MDTS3, molybdopterin cofactor synthesis protein 3 (MOCS3/MoaB); 6PTPS, 6-pyruvoyltetrahydropterin synthase; SPR, sepiapterin reductase; DHPR, 6,7-dihydropteridine reductase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; TS, thymidylate synthase; MTHD, methylenetetrahydrofolate dehydrogenase; MTHF-CH, methenyl-tetrahydrofolate cyclohydrolase; SHMT, serine hydroxymethyltransferase; DHFS, dihydrofolate synthase; THFS, tetrahydro-folylpolyglutamate synthase; Met-tRNA, methionyl-tRNA formyl-transferase.

      Heme

      Heme is an essential cofactor required for the function of various enzymes, including cytochromes, catalases, peroxidases, hemoglobin, and others. Heme alternates between an oxidized and reduced state, enabling heme-containing enzymes to catalyze electron transfer reactions in the ETC and other pathways. Heme can be synthesized de novo, via a highly conserved eight-step pathway (
      • Hamza I.
      • Dailey H.A.
      One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.
      ,
      • Kořený L.
      • Oborník M.
      • Lukeš J.
      Make it, take it, or leave it: heme metabolism of parasites.
      ). Alternatively, it can be salvaged via heme-binding proteins and porphyrin transporters, which have been partially identified in protozoan parasites such as trypanosomes but remain elusive in apicomplexans (
      • Kořený L.
      • Oborník M.
      • Lukeš J.
      Make it, take it, or leave it: heme metabolism of parasites.
      ,
      • Huynh C.
      • Yuan X.
      • Miguel D.C.
      • Renberg R.L.
      • Protchenko O.
      • Philpott C.C.
      • Hamza I.
      • Andrews N.W.
      Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.
      ,
      • Cabello-Donayre M.
      • Orrego L.M.
      • Herráez E.
      • Vargas P.
      • Martínez-García M.
      • Campos-Salinas J.
      • Pérez-Victoria I.
      • Vicente B.
      • Marín J.J.G.
      • Pérez-Victoria J.M.
      Leishmania heme uptake involves LmFLVCRb, a novel porphyrin transporter essential for the parasite.
      ). Whereas Trypanosoma cruzi and Trypanosoma brucei are unable to synthesize heme, Leishmania spp. have acquired the last three enzymes of the biosynthesis pathway via horizontal gene transfer, possibly acquiring and converting heme precursors from the host (
      • Kořený L.
      • Oborník M.
      • Lukeš J.
      Make it, take it, or leave it: heme metabolism of parasites.
      ,
      • Koŕený L.
      • Lukeš J.
      • Oborník M.
      Evolution of the haem synthetic pathway in kinetoplastid flagellates: an essential pathway that is not essential after all?.
      ,
      • Tripodi K.E.J.
      • Menendez Bravo S.M.
      • Cricco J.A.
      Role of heme and heme-proteins in trypanosomatid essential metabolic pathways.
      ). Within the Apicomplexa, Cryptosporidia have lost all enzymes required for heme synthesis, relying entirely on an uptake mechanism. Coccidians and hemosporidians encode all enzymes necessary for de novo synthesis of heme (
      • Kořený L.
      • Oborník M.
      • Lukeš J.
      Make it, take it, or leave it: heme metabolism of parasites.
      ). They possess a peculiar synthesis pathway, which spans three subcellular compartments, the mitochondrion, apicoplast, and cytosol, and comprises enzymes with distinct ancestral origins (
      • Kořený L.
      • Oborník M.
      • Lukeš J.
      Make it, take it, or leave it: heme metabolism of parasites.
      ,
      • Oborník M.
      • Green B.R.
      Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes.
      ) (Fig. 7). The parasites utilize the so-called C4 pathway of α-proteobacterial origin, in which the heme precursor δ-aminolevulinic acid (ALA) is synthesized through condensation of succinyl-CoA and glycine in the mitochondrion. δ-ALA is transported to the apicoplast, where the four-step conversion into coproporphyrinogen III occurs, catalyzed by enzymes originating from the algal endosymbiont (
      • Oborník M.
      • Green B.R.
      Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes.
      ,
      • Koreny L.
      • Sobotka R.
      • Janouskovec J.
      • Keeling P.J.
      • Oborník M.
      Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites.
      ). Coproporphyrinogen III is exported from the apicoplast to the cytosol, where it is converted to protoporphyrinogen IX by a coproporphyrinogen III oxidase (CPO). Protoporphyrinogen IX is subsequently transported to the mitochondrion and converted to heme through the activity of protoporphyrinogen oxidase and ferrochelatase (FC). The contribution of heme uptake versus its de novo synthesis has been investigated in depth in Plasmodium spp. In its blood stages, Plasmodium parasites deal with very high levels of heme, which are released during the digestion of hemoglobin. P. falciparum detoxifies heme by depositing it in a large crystalline pigment termed hemozoin. Hemozoin formation is mediated by a multiprotein complex in the food vacuole, which contains several proteases and a heme detoxification protein (
      • Chugh M.
      • Sundararaman V.
      • Kumar S.
      • Reddy V.S.
      • Siddiqui W.A.
      • Stuart K.D.
      • Malhotra P.
      Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum.
      ). Whereas protein-driven hemozoin formation has been postulated before (
      • Sullivan Jr., D.J.
      • Gluzman I.Y.
      • Goldberg D.E.
      Plasmodium hemozoin formation mediated by histidine-rich proteins.
      ), lipid-driven mechanisms (
      • Bendrat K.
      • Berger B.J.
      • Cerami A.
      Haem polymerization in malaria.
      ,
      • Fitch C.D.
      • Cai G.Z.
      • Chen Y.-F.
      • Shoemaker J.D.
      Involvement of lipids in ferriprotoporphyrin IX polymerization in malaria.
      ) and an autocatalytic process have also been proposed (
      • Dorn A.
      • Stoffel R.
      • Matile H.
      • Bubendorf A.
      • Ridley R.G.
      Malarial haemozoin/β-haematin supports haem polymerization in the absence of protein.
      ). Unsurprisingly, heme synthesis is not essential for Plasmodium during the intraerythrocytic development, but the pathway becomes fitness-conferring during liver stages and is essential for development in the mosquito (
      • Nagaraj V.A.
      • Sundaram B.
      • Varadarajan N.M.
      • Subramani P.A.
      • Kalappa D.M.
      • Ghosh S.K.
      • Padmanaban G.
      Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.
      ,
      • Ke H.
      • Sigala P.A.
      • Miura K.
      • Morrisey J.M.
      • Mather M.W.
      • Crowley J.R.
      • Henderson J.P.
      • Goldberg D.E.
      • Long C.A.
      • Vaidya A.B.
      The heme biosynthesis pathway is essential for Plasmodium falciparum development in mosquito stage but not in blood stages.
      ,
      • Rizopoulos Z.
      • Matuschewski K.
      • Haussig J.M.
      Distinct prominent roles for enzymes of Plasmodium berghei heme biosynthesis in sporozoite and liver stage maturation.
      ,
      • Goldberg D.E.
      • Sigala P.A.
      Plasmodium heme biosynthesis: to be or not to be essential?.
      ,
      • Sigala P.A.
      • Crowley J.R.
      • Henderson J.P.
      • Goldberg D.E.
      Deconvoluting heme biosynthesis to target blood-stage malaria parasites.
      ). Specifically, the loss of FC impairs male gamete formation and ablates oocyst formation in mosquitoes, indicating that Plasmodium can utilize salvaged heme but relies on its synthesis when levels of exogeneous heme become limiting within the insect vector (
      • Nagaraj V.A.
      • Sundaram B.
      • Varadarajan N.M.
      • Subramani P.A.
      • Kalappa D.M.
      • Ghosh S.K.
      • Padmanaban G.
      Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.
      ,
      • Ke H.
      • Sigala P.A.
      • Miura K.
      • Morrisey J.M.
      • Mather M.W.
      • Crowley J.R.
      • Henderson J.P.
      • Goldberg D.E.
      • Long C.A.
      • Vaidya A.B.
      The heme biosynthesis pathway is essential for Plasmodium falciparum development in mosquito stage but not in blood stages.
      ).
      Figure thumbnail gr7
      Figure 7Heme biosynthesis pathway. T. gondii can de novo–synthesize heme in a complex pathway, compartmentalized between the mitochondrion, cytosol, and apicoplast. FS for the enzymes are color-coded (in circles). Experimentally validated enzymes are circled in black.3 ALAS, aminolevulinate synthase; ALAD, aminolevulinate dehydratase; PBGD, porphobilinogen deaminase; UROS, uroporphyrinogen synthase; UROD, uroporphyrinogen decarboxylase; PPO, protoporphyrinogen oxidase.
      Heme has also been intensely researched for its role in determining sensitivity of the parasite to the antimalarial drug artemisinin. Heme-bound iron derived from de novo synthesis or hemoglobin digestion reacts with artemisinin, forming active cytotoxic artemisinin radicals (
      • Meshnick S.R.
      • Thomas A.
      • Ranz A.
      • Xu C.M.
      • Pan H.Z.
      Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action.
      ,
      • Tilley L.
      • Straimer J.
      • Gnädig N.F.
      • Ralph S.A.
      • Fidock D.A.
      Artemisinin action and resistance in Plasmodium falciparum.
      ,
      • Wang J.
      • Zhang C.-J.
      • Chia W.N.
      • Loh C.C.Y.
      • Li Z.
      • Lee Y.M.
      • He Y.
      • Yuan L.-X.
      • Lim T.K.
      • Liu M.
      • Liew C.X.
      • Lee Y.Q.
      • Zhang J.
      • Lu N.
      • Lim C.T.
      • et al.
      Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum.
      ). It has been shown that enhancing heme synthesis, by providing excess heme precursors, increases the sensitivity of Plasmodium to artemisinin. Conversely, the reduction of heme synthesis by genetic means or through pharmacological inhibition decreases sensitivity of both T. gondii and P. falciparum to artemisinin (
      • Zhang S.
      • Gerhard G.S.
      Heme mediates cytotoxicity from artemisinin and serves as a general anti-proliferation target.
      ,
      • Harding C.R.
      • Sidik S.M.
      • Petrova B.
      • Gnädig N.F.
      • Okombo J.
      • Ward K.E.
      • Markus B.M.
      • Fidock D.A.
      • Lourido S.
      Genetic screens reveal a central role for heme biosynthesis in artemisinin susceptibility.
      ).
      Whereas T. gondii does not have to deal with copious amounts of heme as in the intra-erythrocytic stage of P. falciparum, it is also expected to encounter varying levels of heme during its complex life cycle. Based on their FS, all enzymes implicated in the heme synthesis pathway appear highly fitness-conferring (
      • Sidik S.M.
      • Huet D.
      • Ganesan S.M.
      • Huynh M.-H.H.
      • Wang T.
      • Nasamu A.S.
      • Thiru P.
      • Saeij J.P.J.
      • Carruthers V.B.
      • Niles J.C.
      • Lourido S.
      A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.
      ) (Fig. 7), indicating that in vitro tachyzoites are unable to scavenge sufficient amounts of heme from their host. The enzyme catalyzing the second step of the pathway, ALA dehydratase or porphobilinogen synthase, has been characterized biochemically (
      • Shanmugam D.
      • Wu B.
      • Ramirez U.
      • Jaffe E.K.
      • Roos D.S.
      Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.
      ). Its crystal structure revealed that the enzyme functions as an octamer in T. gondii and does not contain any metal ions in the active site, although Mg2+ ions are present at the intersections between pro-octamer dimers (
      • Jaffe E.K.
      • Shanmugam D.
      • Gardberg A.
      • Dieterich S.
      • Sankaran B.
      • Stewart L.J.
      • Myler P.J.
      • Roos D.S.
      Crystal structure of Toxoplasma gondii porphobilinogen synthase.
      ). This metal-independent catalysis is unique to apicomplexans and could render the enzyme an attractive target for intervention.
      Interestingly, T. gondii also encodes two putative and distinct types of coproporphyrinogen oxidases, a CPO and a bacterial-type coproporphyrinogen III dehydrogenase (CPDH) (
      • Layer G.
      • Moser J.
      • Heinz D.W.
      • Jahn D.
      • Schubert W.-D.
      Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes.
      ,
      • Choby J.E.
      • Skaar E.P.
      Heme synthesis and acquisition in bacterial pathogens.
      ). Whereas CPO appears to be highly fitness-conferring based on its FS (−4.64), the oxygen-independent CPDH (+2.29) is the only dispensable enzyme associated with the pathway. Its role in the heme synthesis of T. gondii is still unknown, as it may function as the active CPO in a life cycle stage where oxygen levels are limiting. Consistent with this, RNA-Seq data revealed a striking stage specificity, with CPDH being more than 2-fold up-regulated in bradyzoites, oocysts, and sporozoites (
      • Hehl A.B.
      • Basso W.U.
      • Lippuner C.
      • Ramakrishnan C.
      • Okoniewski M.
      • Walker R.A.
      • Grigg M.E.
      • Smith N.C.
      • Deplazes P.
      Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.
      ). The role of both enzymes was recently investigated through the generation and characterization of mutant parasites lacking CPO, CPDH, or both. The results confirmed that in the absence of CPO, parasites are severely impaired in their cell division and the overall lytic cycle is compromised. Contrastingly, parasites lacking CPDH grow normally as tachyzoites and are not affected in stage conversion to bradyzoites or in cyst formation in mice.3 Furthermore, no aggravation of the phenotype was observed in parasites lacking both enzymes, CPO and CPDH. Overexpression of CPDH in parasites lacking CPO further confirmed a lack of compensation, possibly due to the differential localization of the two enzymes (CPDH in the mitochondrion and CPO in the cytosol).3 Together, these findings indicate that CPDH is dispensable for both tachyzoites and bradyzoites, highlighting that oxygen levels at these stages are sufficient for the oxygen-dependent CPO to function. Importantly, the activity of CPDH has to date not been formally demonstrated, and misannotations of SAM-dependent enzymes have been reported previously (
      • Dailey H.A.
      • Gerdes S.
      • Dailey T.A.
      • Burch J.S.
      • Phillips J.D.
      Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin.
      ). Hence, it remains unclear whether the enzyme truly functions as a CPDH in sporozoites, oocysts, or gametes or whether it functions in a different pathway.
      Importantly, although parasites lacking CPO were severely impaired, they remained viable. On the other hand, depletion of the final enzyme, FC, was not tolerated. Mass spectrometry and fluorescence analyses revealed that cells lacking CPO have 10-fold lower heme levels than WT parasites, but 10-fold higher levels of its precursor protoporphyrin IX (ProtoIX).3 These findings indicate that T. gondii likely does not salvage heme itself but rather its precursors ProtoIX or protoporphyrinogen IX from its host. Hence, FC is absolutely essential for the integration of iron into ProtoIX. Conversion of salvaged ProtoIX or protoporphyrinogen IX to heme appears to be inefficient, leading to the described phenotype. This was further supported by the observation that δ-ALA supplementation rescues the growth defect of T. gondii lacking CPO. δ-ALA supplementation leads to a drastic increase in host ProtoIX levels, probably boosting its uptake by T. gondii and allowing it to restore heme levels.
      In parasites lacking CPO, the lack of heme and accumulation of its precursor are expected to cause deleterious impacts on T. gondii metabolism and development. Heme is crucial for multiple cellular processes; most notably, it serves as an essential cofactor in several enzymes of the ETC, including cytochrome bc1 of complex III, soluble cytochrome c, and the Cox I subunit of Complex IV (
      • van Dooren G.G.
      • Stimmler L.M.
      • McFadden G.I.
      Metabolic maps and functions of the Plasmodium mitochondrion.
      ). It has been proposed that oxidative phosphorylation is the main energy source of tachyzoites and accounts for >90% of the ATP generated in egressed tachyzoites (
      • MacRae J.I.I.
      • Sheiner L.
      • Nahid A.
      • Tonkin C.
      • Striepen B.
      • McConville M.J.J.
      Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii.
      ). We found that heme depletion in parasites lacking CPO largely disables mitochondrial respiration, although residual low levels of respiration were detected, and parasites devoid of CPO remained sensitive to atovaquone treatment, which inhibits the cytochrome bc1 complex of the ETC.3 Strikingly, these parasites appear to survive through markedly increased rates of glycolysis and are unable to survive in the absence of glucose. These observations highlight the importance of de novo heme synthesis in T. gondii but also demonstrate its astonishing flexibility to adapt and survive solely on an inefficient precursor salvage pathway and rewiring its central carbon metabolism. Given the absence of the heme biosynthesis pathway in Cryptosporidia and piroplasms, future research should focus on the identification of heme or hemoprotein transport mechanisms.

      Lipoate

      Lipoate, or lipoic acid, is an essential cofactor and, in most eukaryotes, is synthesized in the mitochondrion and transported to other subcellular compartments. In apicomplexans, at least four metabolic complexes use the lipoic acid as a cofactor: PDH, which resides in the apicoplast (
      • Fleige T.
      • Fischer K.
      • Ferguson D.J.P.
      • Gross U.
      • Bohne W.
      Carbohydrate metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator.
      ), as well as the α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and glycine cleavage complex, which reside in the mitochondrion (
      • Oppenheim R.D.
      • Creek D.J.
      • Macrae J.I.
      • Modrzynska K.K.
      • Pino P.
      • Limenitakis J.
      • Polonais V.
      • Seeber F.
      • Barrett M.P.
      • Billker O.
      • McConville M.J.
      • Soldati-Favre D.
      BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondiiPlasmodium berghei.
      ,
      • Read M.
      • Müller I.B.
      • Mitchell S.L.
      • Sims P.F.
      • Hyde J.E.
      Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum.
      ). The coccidians and hemosporidians are able to synthesize and scavenge lipoic acid, whereas the pathways are absent in Cryptosporidia and Piroplasmida. Unlike plants, which have two isoenzymes, LipA and LipB, for lipoylation in the chloroplast and mitochondria, respectively, apicomplexan genomes encode LipA and LipB. Both enzymes are localized to the apicoplast, and a second enzyme, LplA, is found in the mitochondrion (
      • Thomsen-Zieger N.
      • Schachtner J.
      • Seeber F.
      Apicomplexan parasites contain a single lipoic acid synthase located in the plastid.
      ). Lipoylation of mitochondrial proteins is dramatically reduced when the parasites are grown in lipoic acid–deficient media without affecting the lipoylation of apicoplast proteins (
      • Crawford M.J.
      • Thomsen-Zieger N.
      • Ray M.
      • Schachtner J.
      • Roos D.S.
      • Seeber F.
      Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast.
      ). Contrastingly, the reduced lipoylation of mitochondrial proteins could be rescued via exogenous supplementation of lipoate in the media, indicating the salvage pathway primarily supplies lipoate for this organelle (
      • Crawford M.J.
      • Thomsen-Zieger N.
      • Ray M.
      • Schachtner J.
      • Roos D.S.
      • Seeber F.
      Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast.
      ). As seen by the FS of the LplA gene (−2.60), mitochondrial lipoylation seems essential, whereas LipA (−0.97) and LipB (−1.74) (
      • Sidik S.M.
      • Huet D.
      • Ganesan S.M.
      • Huynh M.-H.H.
      • Wang T.
      • Nasamu A.S.
      • Thiru P.
      • Saeij J.P.J.
      • Carruthers V.B.
      • Niles J.C.
      • Lourido S.
      A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.
      ) in the apicoplast seem dispensable. In the absence of a lipoylated PDH complex, the parasites likely compensate by taking up fatty acids from the host (Fig. 8). Similar observations were reported during the intraerythrocytic stage of P. falciparum (
      • Falkard B.
      • Kumar T.R.S.
      • Hecht L.-S.
      • Matthews K.A.
      • Henrich P.P.
      • Gulati S.
      • Lewis R.E.
      • Manary M.J.
      • Winzeler E.A.
      • Sinnis P.
      • Prigge S.T.
      • Heussler V.
      • Deschermeier C.
      • Fidock D.
      A key role for lipoic acid synthesis during Plasmodium liver stage development.
      ,
      • Günther S.
      • Matuschewski K.
      • Müller S.
      Knockout studies reveal an important role of Plasmodium lipoic acid protein ligase A1 for asexual blood stage parasite survival.
      ). The plasma membrane and organellar transporters involved in lipoate salvage have not yet been identified. It is plausible that lipoate is directly scavenged from the host mitochondria, which is in close contact with the parasitophorous vacuole (
      • Sinai A.P.
      • Webster P.
      • Joiner K.A.
      Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction.
      ).
      Figure thumbnail gr8
      Figure 8Lipoic acid biosynthesis. T. gondii can de novo–synthesize lipoic acid in the apicoplast but also scavenge the metabolite from its host for its requirement within the mitochondrion. The bifunctional LPL enzyme (in blue) utilizes the scavenged lipoate for the posttranslational modification of branched-chain keto-acid dehydrogenase. LipB and LipA generate lipoate for the modification of the E2 subunit of the apicoplast-resident PDH complex. FS for the enzymes are color-coded (in circles). LipB, lipoyl (octanoyl)-ACP-protein N-lipoyl (octanoyl) transferase; LPL, lipoate-protein ligase; LipA, lipoic acid synthase; KADH, branched-chain keto-acid dehydrogenase.

      Shikimate

      Shikimate is an important metabolite found in bacteria, plants, and fungi but is absent in animals. It is important for several biosynthetic processes, including the biosynthesis of folate, aromatic amino acids, and ubiquinone. Shikimate is primarily synthesized from erythrose 4-phosphate and phosphoenolpyruvate and subsequently converted to chorismate in a seven-step reaction. Steps 2–6 for chorismate biosynthesis are carried out by a pentafunctional protein (Fig. 9). In most apicomplexans, including the coccidians, hemosporidians, and Cryptosporidia, a single gene of fungal origin exists, called the AROM complex, encoding for all five activities in a single large polypeptide (
      • Roberts F.
      • Roberts C.W.
      • Johnson J.J.
      • Kyle D.E.
      • Krell T.
      • Coggins J.R.
      • Coombs G.H.
      • Milhous W.K.
      • Tzipori S.
      • Ferguson D.J.P.
      • Chakrabarti D.
      • McLeod R.
      Evidence for the shikimate pathway in apicomplexan parasites.
      ,
      • Peek J.
      • Castiglione G.
      • Shi T.
      • Christendat D.
      Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex.
      ). The presence of all functional domains in T. gondii has been verified with bioinformatic analyses (
      • Campbell S.A.
      • Richards T.A.
      • Mui E.J.
      • Samuel B.U.
      • Coggins J.R.
      • McLeod R.
      • Roberts C.W.
      A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation.
      ,
      • Richards T.A.
      • Dacks J.B.
      • Campbell S.A.
      • Blanchard J.L.
      • Foster P.G.
      • Mcleod R.
      • Roberts C.W.
      Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements.
      ), although in P. falciparum the sequence similarity to the yeast homolog could not be verified for the first two enzymatic activities. However, evidence for the presence of a shikimate pathway was supported in both T. gondii tachyzoites and the erythrocytic stage of P. falciparum, by treating the parasites with the herbicide glyphosate, inhibitor of the 5-enolpyruvylshikimate-3-phosphate synthase, resulting in a growth defect (
      • Schönbrunn E.
      • Eschenburg S.
      • Shuttleworth W.A.
      • Schloss J.V.
      • Amrhein N.
      • Evans J.N.
      • Kabsch W.
      Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.
      ,
      • Roberts C.W.
      • Roberts F.
      • Lyons R.E.
      • Kirisits M.J.
      • Mui E.J.
      • Finnerty J.
      • Johnson J.J.
      • Ferguson D.J.P.
      • Coggins J.R.
      • Krell T.
      • Coombs G.H.
      • Milhous W.K.
      • Kyle D.E.
      • Tzipori S.
      • Barnwell J.
      • et al.
      The shikimate pathway and its branches in apicomplexan parasites.
      ,
      • Keeling P.J.
      • Palmer J.D.
      • Donald R.G.K.
      • Roos D.S.
      • Waller R.F.
      • McFadden G.I.
      Shikimate pathway in apicomplexan parasites.
      ). The effect was reversible with the addition of pABA or folate in the media, suggesting an essential role of shikimate in providing precursors for the biosynthesis of folates (
      • Roberts C.W.
      • Roberts F.
      • Lyons R.E.
      • Kirisits M.J.
      • Mui E.J.
      • Finnerty J.
      • Johnson J.J.
      • Ferguson D.J.P.
      • Coggins J.R.
      • Krell T.
      • Coombs G.H.
      • Milhous W.K.
      • Kyle D.E.
      • Tzipori S.
      • Barnwell J.
      • et al.
      The shikimate pathway and its branches in apicomplexan parasites.
      ). The role of chorismate in folate biosynthesis has been demonstrated in several studies, but its importance for ubiquinone biosynthesis has not been fully defined. Further, the high negative FS of all enzymes involved in the pathway confirms its essentiality for in vitro T. gondii tachyzoites (AROM complex, −5.22; chorismate synthase, −2.84) and could be targeted for intervention against the coccidians and hemosporidians.
      Figure thumbnail gr9
      Figure 9Shikimate, chorismate, and ubiquinone biosynthesis pathway. T. gondii can de novo–synthesize shikimate and chorismate via a pentafunctional AROM complex, catalyzing the initial five steps (shaded in light blue) and chorismate synthase respectively. Chorismate is a precursor for the biosynthesis of ubiquinone, and the FS for the enzymes are color-coded (in circles). 3DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; 5-EPS, 5-enolpyruvylshikimate-3-phosphate; 3-DHQ, 3-dehydroquinate; ADCS, aminodeoxychorismate synthase; ADCL, aminodeoxychorismate lyase; PEP, phosphoenolpyruvate; 4-HB, 4-hydroxybenzoate.

      Ubiquinone

      Ubiquinone, also known as coenzyme Q, is an integral component of the electron transport chain for the transfer of electrons from NADH dehydrogenase (complex I) and succinate dehydrogenase (complex II) to cytochrome bc1 complex (complex III). In most organisms, ubiquinone is synthesized from chorismate in nine enzymatic steps. Most of the pathway is conserved among all apicomplexans, with two enzymes, oxo-acid lyase and 3-octaprenyl-4-hydroxybenzoate carboxy-lyase, missing from the genome, based on bioinformatic approaches. The divergence of these enzymes cannot be ruled out, because a functional synthesis pathway in P. falciparum was shown by detecting differences in the ubiquinone side chains when compared with the host (
      • de Macedo C.S.
      • Uhrig M.L.
      • Kimura E.A.
      • Katzin A.M.
      Characterization of the isoprenoid chain of coenzyme Q in Plasmodium falciparum.
      ). The 4-hydroxybenzoate backbone of ubiquinone receives an isoprenoid side chain via the 4-HB-prenyl-transferase, which has been well-characterized in P. falciparum, and localized to the apicoplast (
      • Tonhosolo R.
      • D'Alexandri F.L.
      • Genta F.A.
      • Wunderlich G.
      • Gozzo F.C.
      • Eberlin M.N.
      • Peres V.J.
      • Kimura E.A.
      • Katzin A.M.
      Identification, molecular cloning and functional characterization of an octaprenyl pyrophosphate synthase in intra-erythrocytic stages of Plasmodium falciparum.
      ). The production of long-chain isoprenoids, however, occurs in the mitochondrion via farnesyl pyrophosphate synthase (
      • Nair S.C.
      • Brooks C.F.
      • Goodman C.D.
      • Sturm A.
      • McFadden G.I.
      • Sundriyal S.
      • Anglin J.L.
      • Song Y.
      • Moreno S.N.J.
      • Striepen B.
      • Striepen B.
      Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii.
      ), which could subsequently be utilized for the synthesis of ubiquinone and other compounds. It was further shown that fosmidomycin, a drug that inhibits the apicoplast-resident isoprenoid biosynthesis pathway, leads to a decline in ubiquinone synthesis (
      • Cassera M.B.
      • Gozzo F.C.
      • D'Alexandri F.L.
      • Merino E.F.
      • del Portillo H.A.
      • Peres V.J.
      • Almeida I.C.
      • Eberlin M.N.
      • Wunderlich G.
      • Wiesner J.
      • Jomaa H.
      • Kimura E.A.
      • Katzin A.M.
      The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.
      ). In T. gondii tachyzoites, the last three steps of the pathway (Fig. 9) display highly negative FS (−3.61, −3.62, and −4.49), highlighting their importance for in vitro proliferation.

      Conclusion

      Apicomplexans possess versatile metabolic capabilities to adapt and adjust to their diverse host environments. Understanding the parasite's requirements for intracellular replication and the contribution of biosynthesis versus uptake of essential metabolites is therefore crucial for the identification of new candidate drug targets (Fig. 10). Whereas the genome sequences of the disease-causing pathogens provide us clues on their metabolic capabilities at a global level, an in-depth understanding of the needs at each life cycle stage is vital. Pathways and enzymes that are essential for proliferation during acute infection may be dispensable upon stage conversion to latency and vice versa. Recent studies encompassing computational, molecular, and metabolomic tools have advanced our understanding of metabolic pathways for the production of key vitamins and cofactors, paving the way for targeted drug development. A few commercially available compounds targeting vitamin and cofactor pathways, such as pyrimethamine and sulfonamides, already exist to treat toxoplasmosis or malaria. With the rise in drug resistance, however, identification of new enzymes absent in the mammalian host may be useful for a target-directed intervention against the apicomplexans.
      Figure thumbnail gr10
      Figure 10List of potential drug targets in the vitamin and cofactor biosynthesis and salvage pathways within a selected class of apicomplexans. Gene IDs for known genes in T. gondii are listed with unknown transporters. Essentialities of the enzymes for known life cycle stages, in vivo conditions, or intracellular organelles are marked in white. A question mark indicates the presence of a biosynthesis enzyme, although its essentiality for a different life cycle stage of the parasite is unknown. Apico, apicoplast; Mito, mitochondrion.

      References

        • Plattner F.
        • Soldati-Favre D.
        Hijacking of host cellular functions by the Apicomplexa.
        Annu. Rev. Microbiol. 2008; 62 (18785844): 471-487
        • Dubey J.P.
        • Lindsay D.S.
        • Speer C.A.
        Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and biology and development of tissue cysts.
        Clin. Microbiol. Rev. 1998; 11 (9564564): 267-299
        • Dubey J.P.
        Long-term persistence of Toxoplasma gondii in tissues of pigs inoculated with T. gondii oocysts and effect of freezing on viability of tissue cysts in pork.
        Am. J. Vet. Res. 1988; 49 (3400928): 910-913
        • Polonais V.
        • Soldati-Favre D.
        Versatility in the acquisition of energy and carbon sources by the Apicomplexa.
        Biol. Cell. 2010; 102 (20586726): 435-445
        • Gold D.A.
        • Kaplan A.D.
        • Lis A.
        • Bett G.C.L.
        • Rosowski E.E.
        • Cirelli K.M.
        • Bougdour A.
        • Sidik S.M.
        • Beck J.R.
        • Lourido S.
        • Egea P.F.
        • Bradley P.J.
        • Hakimi M.-A.
        • Rasmusson R.L.
        • Saeij J.P.J.
        The toxoplasma dense granule proteins GRA17 and GRA23 mediate the movement of small molecules between the host and the parasitophorous vacuole.
        Cell Host Microbe. 2015; 17 (25974303): 642-652
        • Garten M.
        • Nasamu A.S.
        • Niles J.C.
        • Zimmerberg J.
        • Goldberg D.E.
        • Beck J.R.
        EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX.
        Nat. Microbiol. 2018; 3 (30150733): 1090-1098
        • Sherling E.S.
        • van Ooij C.
        Host cell remodeling by pathogens: the exomembrane system in Plasmodium-infected erythrocytes.
        FEMS Microbiol. Rev. 2016; 40 (27587718): 701-721
        • Baumeister S.
        • Winterberg M.
        • Duranton C.
        • Huber S.M.
        • Lang F.
        • Kirk K.
        • Lingelbach K.
        Evidence for the involvement of Plasmodium falciparum proteins in the formation of new permeability pathways in the erythrocyte membrane.
        Mol. Microbiol. 2006; 60 (16573697): 493-504
        • Mehlhorn H.
        • Shein E.
        The piroplasms: life cycle and sexual stages.
        Adv. Parasitol. 1984; 23 (6442536): 37-103
        • Jalovecka M.
        • Hajdusek O.
        • Sojka D.
        • Kopacek P.
        • Malandrin L.
        The complexity of piroplasms life cycles.
        Front. Cell. Infect. Microbiol. 2018; 8 (30083518): 248
        • O'Hara S.P.
        • Chen X.-M.
        The cell biology of Cryptosporidium infection.
        Microbes Infect. 2011; 13 (21458585): 721-730
        • Moore R.B.
        • Oborník M.
        • Janouškovec J.
        • Chrudimský T.
        • Vancová M.
        • Green D.H.
        • Wright S.W.
        • Davies N.W.
        • Bolch C.J.S.
        • Heimann K.
        • Šlapeta
        • Hoegh-Guldberg J.O.
        • Logsdon J.M.
        • Carter D.A.
        A photosynthetic alveolate closely related to apicomplexan parasites.
        Nature. 2008; 451 (18288187): 959-963
        • Song C.
        • Chiasson M.A.
        • Nursimulu N.
        • Hung S.S.
        • Wasmuth J.
        • Grigg M.E.
        • Parkinson J.
        Metabolic reconstruction identifies strain-specific regulation of virulence in Toxoplasma gondii.
        Mol. Syst. Biol. 2013; 9 (24247825): 708
        • Chiappino-Pepe A.
        • Tymoshenko S.
        • Ataman M.
        • Soldati-Favre D.
        • Hatzimanikatis V.
        Bioenergetics-based modeling of Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks.
        PLoS Comput. Biol. 2017; 13 (28333921): e1005397
        • Tymoshenko S.
        • Oppenheim R.D.
        • Agren R.
        • Nielsen J.
        • Soldati-Favre D.
        • Hatzimanikatis V.
        Metabolic needs and capabilities of Toxoplasma gondii through combined computational and experimental analysis.
        PLoS Comput. Biol. 2015; 11 (26001086): e1004261
        • Stanway R.R.
        • Bushell E.
        • Chiappino-Pepe A.
        • Roques M.
        • Sanderson T.
        • Franke-Fayard B.
        • Caldelari R.
        • Golomingi M.
        • Nyonda M.
        • Pandey V.
        • Schwach F.
        • Chevalley S.
        • Ramesar J.
        • Metcalf T.
        • Herd C.
        • et al.
        Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.
        Cell. 2019; 179 (31730853): 1112-1128
        • Sidik S.M.
        • Huet D.
        • Ganesan S.M.
        • Huynh M.-H.H.
        • Wang T.
        • Nasamu A.S.
        • Thiru P.
        • Saeij J.P.J.
        • Carruthers V.B.
        • Niles J.C.
        • Lourido S.
        A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.
        Cell. 2016; 166 (27594426): 1423-1435.e12
        • Zhang M.
        • Wang C.
        • Otto T.D.
        • Oberstaller J.
        • Liao X.
        • Adapa S.R.
        • Udenze K.
        • Bronner I.F.
        • Casandra D.
        • Mayho M.
        • Brown J.
        • Li S.
        • Swanson J.
        • Rayner J.C.
        • Jiang R.H.Y.
        • Adams J.H.
        Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis.
        Science. 2018; 360 (29724925): eaap7847
        • Bushell E.
        • Gomes A.R.
        • Sanderson T.
        • Anar B.
        • Girling G.
        • Herd C.
        • Metcalf T.
        • Modrzynska K.
        • Schwach F.
        • Martin R.E.
        • Mather M.W.
        • McFadden G.I.
        • Parts L.
        • Rutledge G.G.
        • Vaidya A.B.
        • et al.
        Functional profiling of a Plasmodium genome reveals an abundance of essential genes.
        Cell. 2017; 170 (28708996): 260-272.e8
        • Hehl A.B.
        • Basso W.U.
        • Lippuner C.
        • Ramakrishnan C.
        • Okoniewski M.
        • Walker R.A.
        • Grigg M.E.
        • Smith N.C.
        • Deplazes P.
        Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes.
        BMC Genomics. 2015; 16 (25757795): 66
        • Otto T.D.
        • Böhme U.
        • Jackson A.P.
        • Hunt M.
        • Franke-Fayard B.
        • Hoeijmakers W.A.M.
        • Religa A.A.
        • Robertson L.
        • Sanders M.
        • Ogun S.A.
        • Cunningham D.
        • Erhart A.
        • Billker O.
        • Khan S.M.
        • Stunnenberg H.G.
        • et al.
        A comprehensive evaluation of rodent malaria parasite genomes and gene expression.
        BMC Biol. 2014; 12 (25359557): 86
        • Caldelari R.
        • Dogga S.
        • Schmid M.W.
        • Franke-Fayard B.
        • Janse C.J.
        • Soldati-Favre D.
        • Heussler V.
        Transcriptome analysis of Plasmodium berghei during exo-erythrocytic development.
        Malar. J. 2019; 18 (31551073): 330
        • MacRae J.I.I.
        • Sheiner L.
        • Nahid A.
        • Tonkin C.
        • Striepen B.
        • McConville M.J.J.
        Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii.
        Cell Host Microbe. 2012; 12 (23159057): 682-692
        • Nitzsche R.
        • Zagoriy V.
        • Lucius R.
        • Gupta N.
        Metabolic cooperation of glucose and glutamine is essential for the lytic cycle of obligate intracellular parasite Toxoplasma gondii.
        J. Biol. Chem. 2016; 291 (26518878): 126-141
        • Shukla A.
        • Olszewski K.L.
        • Llinás M.
        • Rommereim L.M.
        • Fox B.A.
        • Bzik D.J.
        • Xia D.
        • Wastling J.
        • Beiting D.
        • Roos D.S.
        • Shanmugam D.
        Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii.
        Int. J. Parasitol. 2018; 48 (30176233): 955-968
        • Blume M.
        • Nitzsche R.
        • Sternberg U.
        • Gerlic M.
        • Masters S.L.
        • Gupta N.
        • McConville M.J.
        A Toxoplasma gondii gluconeogenic enzyme contributes to robust central carbon metabolism and is essential for replication and virulence.
        Cell Host Microbe. 2015; 18 (26269956): 210-220
        • Oppenheim R.D.
        • Creek D.J.
        • Macrae J.I.
        • Modrzynska K.K.
        • Pino P.
        • Limenitakis J.
        • Polonais V.
        • Seeber F.
        • Barrett M.P.
        • Billker O.
        • McConville M.J.
        • Soldati-Favre D.
        BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondiiPlasmodium berghei.
        PLoS Pathog. 2014; 10 (25032958): e1004263
        • Ramakrishnan S.
        • Docampo M.D.
        • Macrae J.I.
        • Pujol F.M.
        • Brooks C.F.
        • van Dooren G.G.
        • Hiltunen J.K.
        • Kastaniotis A.J.
        • McConville M.J.
        • Striepen B.
        • Kalervo J.H.
        • Kastaniotis A.J.
        • McConville M.J.
        • Striepen B.
        Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii.
        J. Biol. Chem. 2012; 287 (22179608): 4957-4971
        • Ramakrishnan S.
        • Docampo M.D.
        • MacRae J.I.
        • Ralton J.E.
        • Rupasinghe T.
        • McConville M.J.
        • Striepen B.
        The intracellular parasite Toxoplasma gondii depends on the synthesis of long-chain and very long-chain unsaturated fatty acids not supplied by the host cell.
        Mol. Microbiol. 2015; 97 (25825226): 64-76
        • Pandey V.
        • Hernandez Gardiol D.
        • Chiappino Pepe A.
        • Hatzimanikatis V.
        TEX-FBA: a constraint-based method for integrating gene expression, thermodynamics, and metabolomics data into genome-scale metabolic models.
        bioRxiv. 2019;
        • Tymoshenko S.
        • Oppenheim R.D.
        • Soldati-Favre D.
        • Hatzimanikatis V.
        Functional genomics of Plasmodium falciparum using metabolic modelling and analysis.
        Brief. Funct. Genomics. 2013; 12 (23793264): 316-327
        • Bender D.A.
        Nutritional Biochemistry of the Vitamins. Cambridge University Press, Cambridge, UK2003: 1-8 (2nd Ed., pp. –)
        • Müller S.
        • Kappes B.
        Vitamin and cofactor biosynthesis pathways in Plasmodium and other apicomplexan parasites.
        Trends Parasitol. 2007; 23 (17276140): 112-121
        • Seeber F.
        • Soldati-Favre D.
        Metabolic pathways in the apicoplast of Apicomplexa.
        Int. Rev. Cell Mol. Biol. 2010; 281 (20460186): 161-228
        • Hung S.S.
        • Parkinson J.
        Post-genomics resources and tools for studying apicomplexan metabolism.
        Trends Parasitol. 2011; 27 (21145790): 131-140
        • Fleige T.
        • Limenitakis J.
        • Soldati-Favre D.
        Apicoplast: keep it or leave it.
        Microbes Infect. 2010; 12 (20083219): 253-262
        • Xu P.
        • Widmer G.
        • Wang Y.
        • Ozaki L.S.
        • Alves J.M.
        • Serrano M.G.
        • Puiu D.
        • Manque P.
        • Akiyoshi D.
        • Mackey A.J.
        • Pearson W.R.
        • Dear P.H.
        • Bankier A.T.
        • Peterson D.L.
        • Abrahamsen M.S.
        • Kapur V.
        • Tzipori S.
        • Buck G.A.
        The genome of Cryptosporidium hominis.
        Nature. 2004; 431 (15510150): 1107-1112
        • Burgess S.L.
        • Gilchrist C.A.
        • Lynn T.C.
        • Petri W.A.
        Parasitic protozoa and interactions with the host intestinal microbiota.
        Infect. Immun. 2017; 85 (28584161): e00101-e00117
        • Hellgren O.
        • Bensch S.
        • Videvall E.
        De novo synthesis of thiamine (vitamin B1) is the ancestral state in Plasmodium parasites—evidence from avian haemosporidians.
        Parasitology. 2018; 145 (29229007): 1084-1089
        • Wrenger C.
        • Eschbach M.-L.
        • Müller I.B.
        • Laun N.P.
        • Begley T.P.
        • Walter R.D.
        Vitamin B1de novo synthesis in the human malaria parasite Plasmodium falciparum depends on external provision of 4-amino-5-hydroxymethyl-2-methylpyrimidine.
        Biol. Chem. 2006; 387 (16497163): 41-51
        • Wrenger C.
        • Knöckel J.
        • Walter R.D.
        • Müller I.B.
        Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?.
        Braz. J. Med. Biol. Res. 2008; 41 (18235965): 82-88
        • Tarun A.S.
        • Baer K.
        • Dumpit R.F.
        • Gray S.
        • Lejarcegui N.
        • Frevert U.
        • Kappe S.H.I.
        Quantitative isolation and in vivo imaging of malaria parasite liver stages.
        Int. J. Parasitol. 2006; 36 (16890231): 1283-1293
        • Chan X.W.A.
        • Wrenger C.
        • Stahl K.
        • Bergmann B.
        • Winterberg M.
        • Müller I.B.
        • Saliba K.J.
        • Muller I.B.
        • Saliba K.J.
        Chemical and genetic validation of thiamine utilization as an antimalarial drug target.
        Nat. Commun. 2013; 4 (23804074): 2060
        • Zilles J.L.
        • Croal L.R.
        • Downs D.M.
        Action of the thiamine antagonist bacimethrin on thiamine biosynthesis.
        J. Bacteriol. 2000; 182 (10986269): 5606-5610
        • Rajgopal A.
        • Edmondnson A.
        • Goldman I.D.
        • Zhao R.
        SLC19A3 encodes a second thiamine transporter ThTr2.
        Biochim. Biophys. Acta. 2001; 1537 (11731220): 175-178
        • Subramanian V.S.
        • Marchant J.S.
        • Parker I.
        • Said H.M.
        Cell biology of the human thiamine transporter-1 (hTHTR1). Intracellular trafficking and membrane targeting mechanisms.
        J. Biol. Chem. 2003; 278 (12454006): 3976-3984
        • Driscoll T.P.
        • Verhoeve V.I.
        • Guillotte M.L.
        • Lehman S.S.
        • Rennoll S.A.
        • Beier-Sexton M.
        • Rahman M.S.
        • Azad A.F.
        • Gillespie J.J.
        Wholly Rickettsia! Reconstructed metabolic profile of the quintessential bacterial parasite of eukaryotic cells.
        MBio. 2017; 8 (28951473): e00817-e00859
        • Zerez C.R.
        • Roth Jr., E.F.
        • Schulman S.
        • Tanaka K.R.
        Increased nicotinamide adenine dinucleotide content and synthesis in Plasmodium falciparum-infected human erythrocytes.
        Blood. 1990; 75 (2183889): 1705-1710
        • Beri D.
        • Ramdani G.
        • Balan B.
        • Gadara D.
        • Poojary M.
        • Momeux L.
        • Tatu U.
        • Langsley G.
        Insights into physiological roles of unique metabolites released from Plasmodium-infected RBCs and their potential as clinical biomarkers for malaria.
        Sci. Rep. 2019; 9 (30814599): 2875
        • O'Hara J.K.
        • Kerwin L.J.
        • Cobbold S.A.
        • Tai J.
        • Bedell T.A.
        • Reider P.J.
        • Llinás M.
        Targeting NAD+ metabolism in the human malaria parasite Plasmodium falciparum.
        PLoS One. 2014; 9 (24747974): e94061
        • Mageed S.N.
        • Cunningham F.
        • Hung A.W.
        • Silvestre H.L.
        • Wen S.
        • Blundell T.L.
        • Abell C.
        • McConkey G.A.
        Pantothenic acid biosynthesis in the parasite Toxoplasma gondii: a target for chemotherapy.
        Antimicrob. Agents Chemother. 2014; 58 (25049241): 6345-6353
        • Ciulli A.
        • Chirgadze D.Y.
        • Smith A.G.
        • Blundell T.L.
        • Abell C.
        Crystal structure of Escherichia coli ketopantoate reductase in a ternary complex with NADP+ and pantoate bound.
        J. Biol. Chem. 2007; 282 (17229734): 8487-8497
        • von Delft F.
        • Inoue T.
        • Saldanha S.A.
        • Ottenhof H.H.
        • Schmitzberger F.
        • Birch L.M.
        • Dhanaraj V.
        • Witty M.
        • Smith A.G.
        • Blundell T.L.
        • Abell C.
        Structure of E. coli ketopantoate hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved by locating 160 selenomethionine sites.
        Structure. 2003; 11 (12906829): 985-996
        • von Delft F.
        • Lewendon A.
        • Dhanaraj V.
        • Blundell T.L.
        • Abell C.
        • Smith A.G.
        The crystal structure of E. coli pantothenate synthetase confirms it as a member of the cytidylyltransferase superfamily.
        Structure. 2001; 9 (11377204): 439-450
        • Saliba K.J.
        • Horner H.A.
        • Kirk K.
        Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum.
        J. Biol. Chem. 1998; 273 (9553068): 10190-10195
        • Augagneur Y.
        • Jaubert L.
        • Schiavoni M.
        • Pachikara N.
        • Garg A.
        • Usmani-Brown S.
        • Wesolowski D.
        • Zeller S.
        • Ghosal A.
        • Cornillot E.
        • Said H.M.
        • Kumar P.
        • Altman S.
        • Ben Mamoun C.
        Identification and functional analysis of the primary pantothenate transporter, PfPAT, of the human malaria parasite Plasmodium falciparum.
        J. Biol. Chem. 2013; 288 (23729665): 20558-20567
        • Kehrer J.
        • Singer M.
        • Lemgruber L.
        • Silva P.A.G.C.
        • Frischknecht F.
        • Mair G.R.
        A putative small solute transporter is responsible for the secretion of G377 and TRAP-containing secretory vesicles during Plasmodium gamete egress and sporozoite motility.
        PLoS Pathog. 2016; 12 (27427910): e1005734
        • Hammoudi P.-M.
        • Maco B.
        • Dogga S.K.
        • Frénal K.
        • Soldati-Favre D.
        Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis.
        Mol. Microbiol. 2018; 109 (29738095): 225-244
        • Pietrocola F.
        • Galluzzi L.
        • Bravo-San Pedro J.M.
        • Madeo F.
        • Kroemer G.
        Acetyl coenzyme A: a central metabolite and second messenger.
        Cell Metab. 2015; 21 (26039447): 805-821
        • Tjhin E.T.
        • Spry C.
        • Sewell A.L.
        • Hoegl A.
        • Barnard L.
        • Sexton A.E.
        • Siddiqui G.
        • Howieson V.M.
        • Maier A.G.
        • Creek D.J.
        • Strauss E.
        • Marquez R.
        • Auclair K.
        • Saliba K.J.
        Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues.
        PLoS Pathog. 2018; 14 (29614109): e1006918
        • de Villiers M.
        • Spry C.
        • Macuamule C.J.
        • Barnard L.
        • Wells G.
        • Saliba K.J.
        • Strauss E.
        Antiplasmodial mode of action of pantothenamides: pantothenate kinase serves as a metabolic activator not as a target.
        ACS Infect. Dis. 2017; 3 (28437604): 527-541
        • Schalkwijk J.
        • Allman E.L.
        • Jansen P.A.
        • Vries L.E
        • de Jackowski S.
        • Botman P.N.
        • Beuckens-Schortinghuis C.A.
        • Koolen K.M.
        • Bolscher J.M.
        • Vos M.W.
        • Miller K.
        • Reeves S.
        • Pett H.
        • Trevitt G.
        • Wittlin S.
        • et al.
        Antimalarial pantothenamide metabolites target acetyl-CoA synthesis in Plasmodium falciparum.
        bioRxiv. 2018;
        • Hart R.J.
        • Abraham A.
        • Aly A.S.I.
        Genetic characterization of coenzyme A biosynthesis reveals essential distinctive functions during malaria parasite development in blood and mosquito.
        Front. Cell. Infect. Microbiol. 2017; 7 (28676844): 260
        • de Villiers M.
        • Barnard L.
        • Koekemoer L.
        • Snoep J.L.
        • Strauss E.
        Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis.
        FEBS J. 2014; 281 (25156889): 4731-4753
        • Srinivasan B.
        • Baratashvili M.
        • van der Zwaag M.
        • Kanon B.
        • Colombelli C.
        • Lambrechts R.A.
        • Schaap O.
        • Nollen E.A.
        • Podgoršek A.
        • Kosec G.
        • Petković H.
        • Hayflick S.
        • Tiranti V.
        • Reijngoud D.-J.
        • Grzeschik N.A.
        • Sibon O.C.M.
        Extracellular 4′-phosphopantetheine is a source for intracellular coenzyme A synthesis.
        Nat. Chem. Biol. 2015; 11 (26322826): 784-792
        • Percudani R.
        • Peracchi A.
        A genomic overview of pyridoxal-phosphate-dependent enzymes.
        EMBO Rep. 2003; 4 (12949584): 850-854
        • Hoegl A.
        • Nodwell M.B.
        • Kirsch V.C.
        • Bach N.C.
        • Pfanzelt M.
        • Stahl M.
        • Schneider S.
        • Sieber S.A.
        Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics.
        Nat. Chem. 2018; 10 (30297752): 1234-1245
        • Fitzpatrick T.B.
        • Amrhein N.
        • Kappes B.
        • Macheroux P.
        • Tews I.
        • Raschle T.
        Two independent routes of de novo vitamin B6 biosynthesis: not that different after all.
        Biochem. J. 2007; 407 (17822383): 1-13
        • Knöckel J.
        • Müller I.B.
        • Bergmann B.
        • Walter R.D.
        • Wrenger C.
        The apicomplexan parasite Toxoplasma gondii generates pyridoxal phosphate de novo.
        Mol. Biochem. Parasitol. 2007; 152 (17222923): 108-111
        • Gengenbacher M.
        • Fitzpatrick T.B.
        • Raschle T.
        • Flicker K.
        • Sinning I.
        • Müller S.
        • Macheroux P.
        • Tews I.
        • Kappes B.
        Vitamin B6 biosynthesis by the malaria parasite Plasmodium falciparum: biochemical and structural insights.
        J. Biol. Chem. 2006; 281 (16339145): 3633-3641
        • Palm D.
        • Klein H.W.
        • Schinzel R.
        • Buehner M.
        • Helmreich E.J.M.
        The role of pyridoxal 5′-phosphate in glycogen phosphorylase catalysis.
        Biochemistry. 1990; 29 (2182117): 1099-1107
        • Sugi T.
        • Tu V.
        • Ma Y.
        • Tomita T.
        • Weiss L.M.
        Toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of brain cysts.
        MBio. 2017; 8 (28851850): e01217-e01289
        • Van de Kamp J.L.
        • Westrick J.A.
        • Smolen A.
        B6 vitamer concentrations in mouse plasma, erythrocytes and tissues.
        Nutr. Res. 1995; 15: 415-422
        • Cassera M.B.
        • Gozzo F.C.
        • D'Alexandri F.L.
        • Merino E.F.
        • del Portillo H.A.
        • Peres V.J.
        • Almeida I.C.
        • Eberlin M.N.
        • Wunderlich G.
        • Wiesner J.
        • Jomaa H.
        • Kimura E.A.
        • Katzin A.M.
        The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.
        J. Biol. Chem. 2004; 279 (15452112): 51749-51759
        • Wrenger C.
        • Eschbach M.-L.
        • Müller I.B.
        • Warnecke D.
        • Walter R.D.
        Analysis of the vitamin B6 biosynthesis pathway in the human malaria parasite Plasmodium falciparum.
        J. Biol. Chem. 2005; 280 (15590634): 5242-5248
        • Müller I.B.
        • Hyde J.E.
        • Wrenger C.
        Vitamin B metabolism in Plasmodium falciparum as a source of drug targets.
        Trends Parasitol. 2010; 26 (19939733): 35-43
        • Knöckel J.
        • Müller I.B.
        • Butzloff S.
        • Bergmann B.
        • Walter R.D.
        • Wrenger C.
        The antioxidative effect of de novo generated vitamin B6 in Plasmodium falciparum validated by protein interference.
        Biochem. J. 2012; 443 (22242896): 397-405
        • Kronenberger T.
        • Lindner J.
        • Meissner K.A.
        • Zimbres F.M.
        • Coronado M.A.
        • Sauer F.M.
        • Schettert I.
        • Wrenger C.
        Vitamin B6-dependent enzymes in the human malaria parasite Plasmodium falciparum: a druggable target?.
        Biomed. Res. Int. 2014; 2014 (24524072): 108516
        • Müller I.B.
        • Wu F.
        • Bergmann B.
        • Knöckel J.
        • Walter R.D.
        • Gehring H.
        • Wrenger C.
        Poisoning pyridoxal 5-phosphate-dependent enzymes: a new strategy to target the malaria parasite Plasmodium falciparum.
        PLoS One. 2009; 4 (19197387): e4406
        • Jelenska J.
        • Crawford M.J.
        • Harb O.S.
        • Zuther E.
        • Haselkorn R.
        • Roos D.S.
        • Gornicki P.
        Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii.
        Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11226307): 2723-2728
        • Chapman-Smith A.
        • Cronan Jr., J.E.
        Molecular biology of biotin attachment to proteins.
        J. Nutr. 1999; 129 (10064313): 477S-484S
        • Beckett D.
        The Escherichia coli biotin regulatory system: a transcriptional switch.
        J. Nutr. Biochem. 2005; 16 (15992680): 411-415
        • Hebbeln P.
        • Rodionov D.A.
        • Alfandega A.
        • Eitinger T.
        Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17301237): 2909-2914
        • Daberkow R.L.
        • White B.R.
        • Cederberg R.A.
        • Griffin J.B.
        • Zempleni J.
        Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells.
        J. Nutr. 2003; 133 (12949353): 2703-2706
        • Massimine K.M.
        • Doan L.T.
        • Atreya C.A.
        • Stedman T.T.
        • Anderson K.S.
        • Joiner K.A.
        • Coppens I.
        Toxoplasma gondii is capable of exogenous folate transport: a likely expansion of the BT1 family of transmembrane proteins.
        Mol. Biochem. Parasitol. 2005; 144 (16159678): 44-54
        • Salcedo-Sora J.E.
        • Ochong E.
        • Beveridge S.
        • Johnson D.
        • Nzila A.
        • Biagini G.A.
        • Stocks P.A.
        • O'Neill P.M.
        • Krishna S.
        • Bray P.G.
        • Ward S.A.
        The molecular basis of folate salvage in Plasmodium falciparum: characterization of two folate transporters.
        J. Biol. Chem. 2011; 286 (21998306): 44659-44668
        • Hyde J.E.
        Exploring the folate pathway in Plasmodium falciparum.
        Acta Trop. 2005; 94 (15845349): 191-206
        • Nzila A.
        • Ward S.A.
        • Marsh K.
        • Sims P.F.G.
        • Hyde J.E.
        Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy.
        Trends Parasitol. 2005; 21 (15922251): 292-298
        • Heinberg A.
        • Kirkman L.
        The molecular basis of antifolate resistance in Plasmodium falciparum: looking beyond point mutations.
        Ann. N.Y. Acad. Sci. 2015; 1342 (25694157): 10-18
        • Matz J.M.
        • Watanabe M.
        • Falade M.
        • Tohge T.
        • Hoefgen R.
        • Matuschewski K.
        Plasmodium para-aminobenzoate synthesis and salvage resolve avoidance of folate competition and adaptation to host diet.
        Cell Rep. 2019; 26 (30625318): 356-363.e4
        • Mather M.W.
        • Ke H.
        para-Aminobenzoate synthesis versus salvage in malaria parasites.
        Trends Parasitol. 2019; 35 (30709568): 176-178
        • Hamza I.
        • Dailey H.A.
        One ring to rule them all: trafficking of heme and heme synthesis intermediates in the metazoans.
        Biochim. Biophys. Acta. 2012; 1823 (22575458): 1617-1632
        • Kořený L.
        • Oborník M.
        • Lukeš J.
        Make it, take it, or leave it: heme metabolism of parasites.
        PLoS Pathog. 2013; 9 (23349629): e1003088
        • Huynh C.
        • Yuan X.
        • Miguel D.C.
        • Renberg R.L.
        • Protchenko O.
        • Philpott C.C.
        • Hamza I.
        • Andrews N.W.
        Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.
        PLoS Pathog. 2012; 8 (22807677): e1002795
        • Cabello-Donayre M.
        • Orrego L.M.
        • Herráez E.
        • Vargas P.
        • Martínez-García M.
        • Campos-Salinas J.
        • Pérez-Victoria I.
        • Vicente B.
        • Marín J.J.G.
        • Pérez-Victoria J.M.
        Leishmania heme uptake involves LmFLVCRb, a novel porphyrin transporter essential for the parasite.
        Cell. Mol. Life Sci. 2019; (31372684)
        • Koŕený L.
        • Lukeš J.
        • Oborník M.
        Evolution of the haem synthetic pathway in kinetoplastid flagellates: an essential pathway that is not essential after all?.
        Int. J. Parasitol. 2010; 40 (19968994): 149-156
        • Tripodi K.E.J.
        • Menendez Bravo S.M.
        • Cricco J.A.
        Role of heme and heme-proteins in trypanosomatid essential metabolic pathways.
        Enzyme Res. 2011; 2011 (21603276): 873230
        • Oborník M.
        • Green B.R.
        Mosaic origin of the heme biosynthesis pathway in photosynthetic eukaryotes.
        Mol. Biol. Evol. 2005; 22 (16093570): 2343-2353
        • Koreny L.
        • Sobotka R.
        • Janouskovec J.
        • Keeling P.J.
        • Oborník M.
        Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites.
        Plant Cell. 2011; 23 (21963666): 3454-3462
        • Chugh M.
        • Sundararaman V.
        • Kumar S.
        • Reddy V.S.
        • Siddiqui W.A.
        • Stuart K.D.
        • Malhotra P.
        Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23471987): 5392-5397
        • Sullivan Jr., D.J.
        • Gluzman I.Y.
        • Goldberg D.E.
        Plasmodium hemozoin formation mediated by histidine-rich proteins.
        Science. 1996; 271 (8539625): 219-222
        • Bendrat K.
        • Berger B.J.
        • Cerami A.
        Haem polymerization in malaria.
        Nature. 1995; 378 (7477315): 138-139
        • Fitch C.D.
        • Cai G.Z.
        • Chen Y.-F.
        • Shoemaker J.D.
        Involvement of lipids in ferriprotoporphyrin IX polymerization in malaria.
        Biochim. Biophys. Acta. 1999; 1454 (10354512): 31-37
        • Dorn A.
        • Stoffel R.
        • Matile H.
        • Bubendorf A.
        • Ridley R.G.
        Malarial haemozoin/β-haematin supports haem polymerization in the absence of protein.
        Nature. 1995; 374 (7885447): 269-271
        • Nagaraj V.A.
        • Sundaram B.
        • Varadarajan N.M.
        • Subramani P.A.
        • Kalappa D.M.
        • Ghosh S.K.
        • Padmanaban G.
        Malaria parasite-synthesized heme is essential in the mosquito and liver stages and complements host heme in the blood stages of infection.
        PLoS Pathog. 2013; 9 (23935500): e1003522
        • Ke H.
        • Sigala P.A.
        • Miura K.
        • Morrisey J.M.
        • Mather M.W.
        • Crowley J.R.
        • Henderson J.P.
        • Goldberg D.E.
        • Long C.A.
        • Vaidya A.B.
        The heme biosynthesis pathway is essential for Plasmodium falciparum development in mosquito stage but not in blood stages.
        J. Biol. Chem. 2014; 289 (25352601): 34827-34837
        • Rizopoulos Z.
        • Matuschewski K.
        • Haussig J.M.
        Distinct prominent roles for enzymes of Plasmodium berghei heme biosynthesis in sporozoite and liver stage maturation.
        Infect. Immun. 2016; 84 (27600503): 3252-3262
        • Goldberg D.E.
        • Sigala P.A.
        Plasmodium heme biosynthesis: to be or not to be essential?.
        PLoS Pathog. 2017; 13 (28957449): e1006511
        • Sigala P.A.
        • Crowley J.R.
        • Henderson J.P.
        • Goldberg D.E.
        Deconvoluting heme biosynthesis to target blood-stage malaria parasites.
        Elife. 2015; (26173178)
        • Meshnick S.R.
        • Thomas A.
        • Ranz A.
        • Xu C.M.
        • Pan H.Z.
        Artemisinin (qinghaosu): the role of intracellular hemin in its mechanism of antimalarial action.
        Mol. Biochem. Parasitol. 1991; 49 (1775162): 181-189
        • Tilley L.
        • Straimer J.
        • Gnädig N.F.
        • Ralph S.A.
        • Fidock D.A.
        Artemisinin action and resistance in Plasmodium falciparum.
        Trends Parasitol. 2016; 32 (27289273): 682-696
        • Wang J.
        • Zhang C.-J.
        • Chia W.N.
        • Loh C.C.Y.
        • Li Z.
        • Lee Y.M.
        • He Y.
        • Yuan L.-X.
        • Lim T.K.
        • Liu M.
        • Liew C.X.
        • Lee Y.Q.
        • Zhang J.
        • Lu N.
        • Lim C.T.
        • et al.
        Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum.
        Nat. Commun. 2015; 6 (26694030): 10111
        • Zhang S.
        • Gerhard G.S.
        Heme mediates cytotoxicity from artemisinin and serves as a general anti-proliferation target.
        PLoS One. 2009; 4 (19862332): e7472
        • Harding C.R.
        • Sidik S.M.
        • Petrova B.
        • Gnädig N.F.
        • Okombo J.
        • Ward K.E.
        • Markus B.M.
        • Fidock D.A.
        • Lourido S.
        Genetic screens reveal a central role for heme biosynthesis in artemisinin susceptibility.
        bioRxiv. 2019;
        • Shanmugam D.
        • Wu B.
        • Ramirez U.
        • Jaffe E.K.
        • Roos D.S.
        Plastid-associated porphobilinogen synthase from Toxoplasma gondii: kinetic and structural properties validate therapeutic potential.
        J. Biol. Chem. 2010; 285 (20442414): 22122-22131
        • Jaffe E.K.
        • Shanmugam D.
        • Gardberg A.
        • Dieterich S.
        • Sankaran B.
        • Stewart L.J.
        • Myler P.J.
        • Roos D.S.
        Crystal structure of Toxoplasma gondii porphobilinogen synthase.
        J. Biol. Chem. 2011; 286 (21383008): 15298-15307
        • Layer G.
        • Moser J.
        • Heinz D.W.
        • Jahn D.
        • Schubert W.-D.
        Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of radical SAM enzymes.
        EMBO J. 2003; 22 (14633981): 6214-6224
        • Choby J.E.
        • Skaar E.P.
        Heme synthesis and acquisition in bacterial pathogens.
        J. Mol. Biol. 2016; 428 (27019298): 3408-3428
        • Dailey H.A.
        • Gerdes S.
        • Dailey T.A.
        • Burch J.S.
        • Phillips J.D.
        Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin.
        Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25646457): 2210-2215
        • van Dooren G.G.
        • Stimmler L.M.
        • McFadden G.I.
        Metabolic maps and functions of the Plasmodium mitochondrion.
        FEMS Microbiol Rev. 2006; 30 (16774588): 596-630
        • Fleige T.
        • Fischer K.
        • Ferguson D.J.P.
        • Gross U.
        • Bohne W.
        Carbohydrate metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator.
        Eukaryot. Cell. 2007; 6 (17449654): 984-996
        • Read M.
        • Müller I.B.
        • Mitchell S.L.
        • Sims P.F.
        • Hyde J.E.
        Dynamic subcellular localization of isoforms of the folate pathway enzyme serine hydroxymethyltransferase (SHMT) through the erythrocytic cycle of Plasmodium falciparum.
        Malar. J. 2010; 9 (21129192): 351
        • Thomsen-Zieger N.
        • Schachtner J.
        • Seeber F.
        Apicomplexan parasites contain a single lipoic acid synthase located in the plastid.
        FEBS Lett. 2003; 547 (12860390): 80-86
        • Crawford M.J.
        • Thomsen-Zieger N.
        • Ray M.
        • Schachtner J.
        • Roos D.S.
        • Seeber F.
        Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast.
        EMBO J. 2006; 25 (16778769): 3214-3222
        • Falkard B.
        • Kumar T.R.S.
        • Hecht L.-S.
        • Matthews K.A.
        • Henrich P.P.
        • Gulati S.
        • Lewis R.E.
        • Manary M.J.
        • Winzeler E.A.
        • Sinnis P.
        • Prigge S.T.
        • Heussler V.
        • Deschermeier C.
        • Fidock D.
        A key role for lipoic acid synthesis during Plasmodium liver stage development.
        Cell Microbiol. 2013; 15 (23490300): 1585-1604
        • Günther S.
        • Matuschewski K.
        • Müller S.
        Knockout studies reveal an important role of Plasmodium lipoic acid protein ligase A1 for asexual blood stage parasite survival.
        PLoS One. 2009; 4 (19434237): e5510
        • Sinai A.P.
        • Webster P.
        • Joiner K.A.
        Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction.
        J. Cell Sci. 1997; 110 (9378762): 2117-2128
        • Roberts F.
        • Roberts C.W.
        • Johnson J.J.
        • Kyle D.E.
        • Krell T.
        • Coggins J.R.
        • Coombs G.H.
        • Milhous W.K.
        • Tzipori S.
        • Ferguson D.J.P.
        • Chakrabarti D.
        • McLeod R.
        Evidence for the shikimate pathway in apicomplexan parasites.
        Nature. 1998; 393 (9655396): 801-805
        • Peek J.
        • Castiglione G.
        • Shi T.
        • Christendat D.
        Isolation and molecular characterization of the shikimate dehydrogenase domain from the Toxoplasma gondii AROM complex.
        Mol. Biochem. Parasitol. 2014; 194 (24731949): 16-19
        • Campbell S.A.
        • Richards T.A.
        • Mui E.J.
        • Samuel B.U.
        • Coggins J.R.
        • McLeod R.
        • Roberts C.W.
        A complete shikimate pathway in Toxoplasma gondii: an ancient eukaryotic innovation.
        Int. J. Parasitol. 2004; 34 (14711585): 5-13
        • Richards T.A.
        • Dacks J.B.
        • Campbell S.A.
        • Blanchard J.L.
        • Foster P.G.
        • Mcleod R.
        • Roberts C.W.
        Evolutionary origins of the eukaryotic shikimate pathway: gene fusions, horizontal gene transfer, and endosymbiotic replacements.
        Eukaryot. Cell. 2006; 5 (16963634): 1517-1531
        • Schönbrunn E.
        • Eschenburg S.
        • Shuttleworth W.A.
        • Schloss J.V.
        • Amrhein N.
        • Evans J.N.
        • Kabsch W.
        Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.
        Proc. Natl. Acad. Sci. U.S.A. 2001; 98 (11171958): 1376-1380
        • Roberts C.W.
        • Roberts F.
        • Lyons R.E.
        • Kirisits M.J.
        • Mui E.J.
        • Finnerty J.
        • Johnson J.J.
        • Ferguson D.J.P.
        • Coggins J.R.
        • Krell T.
        • Coombs G.H.
        • Milhous W.K.
        • Kyle D.E.
        • Tzipori S.
        • Barnwell J.
        • et al.
        The shikimate pathway and its branches in apicomplexan parasites.
        J. Infect. Dis. 2002; 185 (11865437): S25-S36
        • Keeling P.J.
        • Palmer J.D.
        • Donald R.G.K.
        • Roos D.S.
        • Waller R.F.
        • McFadden G.I.
        Shikimate pathway in apicomplexan parasites.
        Nature. 1999; 397 (9930696): 219-220
        • de Macedo C.S.
        • Uhrig M.L.
        • Kimura E.A.
        • Katzin A.M.
        Characterization of the isoprenoid chain of coenzyme Q in Plasmodium falciparum.
        FEMS Microbiol. Lett. 2002; 207 (11886744): 13-20
        • Tonhosolo R.
        • D'Alexandri F.L.
        • Genta F.A.
        • Wunderlich G.
        • Gozzo F.C.
        • Eberlin M.N.
        • Peres V.J.
        • Kimura E.A.
        • Katzin A.M.
        Identification, molecular cloning and functional characterization of an octaprenyl pyrophosphate synthase in intra-erythrocytic stages of Plasmodium falciparum.
        Biochem. J. 2005; 392 (15984931): 117-126
        • Nair S.C.
        • Brooks C.F.
        • Goodman C.D.
        • Sturm A.
        • McFadden G.I.
        • Sundriyal S.
        • Anglin J.L.
        • Song Y.
        • Moreno S.N.J.
        • Striepen B.
        • Striepen B.
        Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii.
        J. Exp. Med. 2011; 208 (21690250): 1547-1559