Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is a ubiquitous, protozoan parasite contributing significantly to global human and animal health. In the host, this obligate intracellular parasite converts into a latent tissue cyst form known as the bradyzoite, which is impervious to the immune response. The tissue cysts facilitate wide-spread transmission through the food chain and give rise to chronic toxoplasmosis in immune compromised patients. In addition, they may reactivate into replicating tachyzoites which cause tissue damage and disseminated disease. Current available drugs do not appear to have appreciable activity against latent bradyzoites. Therefore, a better understanding of the molecular mechanisms that drive interconversion between tachyzoite and bradyzoite forms is required to manage transmission and pathogenesis of Toxoplasma. Conversion to the bradyzoite is accompanied by an altered transcriptome, but the molecular players directing this process are largely uncharacterized. Studies of stage-specific promoters revealed that conventional cis-acting mechanisms operate to regulate developmental gene expression during tissue cyst formation. The major class of transcription factor likely to work through these cis-regulatory elements appears to be related to the Apetala-2 (AP2) family in plants. The Toxoplasma genome contains nearly 70 proteins harboring at least one predicted AP2 domain, but to date only three of these T. gondii AP2 proteins have been linked to bradyzoite development. We show that the putative T. gondii transcription factor, AP2IX-4, is localized to the parasite nucleus and exclusively expressed in tachyzoites and bradyzoites undergoing division. Knockout of AP2IX-4 had negligible effect on tachyzoite replication, but resulted in a reduced frequency of bradyzoite cysts in response to alkaline stress induction – a defect that is reversible by complementation. Microarray analyses revealed an enhanced activation of bradyzoite-associated genes in the AP2IX-4 knockout during alkaline conditions. In mice, the loss of AP2IX-4 resulted in a modest virulence defect and reduced brain cyst burden. Complementation of the AP2IX-4 knockout restored cyst counts to wild-type levels. These findings illustrate the complex role of AP2IX-4 in bradyzoite development and that certain transcriptional mechanisms responsible for tissue cyst development operate across parasite division.

Indiana University-Purdue University Indianapolis (IUPUI) / GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones.

Toxoplasma gondii ist ein obligater intrazellulärer Parasit, der bei Menschen und Nutztieren Toxoplasmose verursacht. Die Phospholipid-Biosynthese ist entscheidend für das erfolgreiche intrazelluläre Überleben und die Replikation des Parasiten, da sie eine wichtige Rolle bei der Biogenese von Membranorganellen, der Signal- transduktion und anderen zellulären Prozessen spielt. Hier untersuchten wir die physiologische Bedeutung von zwei Synthesewegen, die für PtdEtn und PtdIns verantwortlich sind. Wir zeigen das Vorhandensein einer neuartigen PtdIns-Synthase (PIS) in T. gondii, die als TgPIS bezeichnet wird und ein funktionelles Enzym mit einem katalytisch wichtigen CDP-Alkohol- Phosphotransferase-Motiv codiert, das sich ausschließlich im Golgi-Apparat befindet. Der Parasit importiert Myoinosit aus dem Milieu und verwendet es zusammen mit de novo synthetisiertem CDP- Diacylglycerin, um PtdIns zu produzieren. Eine durch Auxin induzierbare bedingte Unterdrückung von TgPIS schaltet den Lysezyklus des Parasiten aufgrund von Defekten in der Replikation, Motilität und Austritt in Säugetierzellen aus. Das Lipidom-Profiling der PIS-Mutante zeigt eine selektive Reduktion bestimmter PtdIns- und PtdThr-Spezies, wohingegen ausgewählte PtdGro-, PtdSer- und BMP-Spezies erhöht sind, was auf eine enge Interregulation und Homöostase von anionischen Phospholipiden zur Aufrechterhaltung der Membranintegrität hindeutet. Zusätzlich identifizierten wir eine Ethanolamin-Cytidyltransferase (TgECT), das geschwindigkeitsbestimmende Enzym des Kennedy-Signalwegs, das im Cytosol lokalisiert ist. Das Enzym ist eindeutig für den Lysezyklus essentiell, da seine genetische Ablation nicht durchführbar ist und der durch Auxin meditierte bedingte Abbau des Proteins das Parasitenwachstum in Plaqueassays stark beeinträchtigt. Die Lipidomanalyse der Mutante identifizierte eine wichtige Rolle bei der Biogenese ausgewählter Arten von PtdEtn, PtdSer und PtdThr. Darüber hinaus haben wir festgestellt, dass TgECT für die Erzeugung von Ethanolamin-Phosphor-Ceramid (EPC) erforderlich ist, einem seltenen Sphingolipid, das nur eine begrenzte Anzahl von Organismen enthalten. / Toxoplasma gondii is an obligate intracellular parasite that causes Toxoplasmosis in human and livestock. Phospholipid biosynthesis is crucial for the successful intracellular survival and replication of the parasites, as the phospholipids have important roles in the biogenesis of membrane organelles, signal transduction and other cellular processes. Here, we dissected the physiological importance of two pathways accounting for the synthesis of PtdEtn and PtdIns. We demonstrated the presence of a novel PtdIns synthase (PIS) in T.gondii termed TgPIS, expressing a functional enzyme with a catalytically vital CDP-alcohol phosphotransferase motif, which resides exclusively in the Golgi body. The parasite imports myo-inositol from milieu, and co-utilizes de novo-synthesized CDP-diacylglycerol to produce PtdIns. An auxin-inducible conditional repression of TgPIS abrogated the lytic cycle of the parasite in mammalian cells due to defects in the replication, motility and egress. Lipidomic profiling of the PIS mutant demonstrated selective reduction of certain PtdIns and PtdThr species, whereas selected PtdGro, PtdSer and BMP species were increased, which suggested a tight inter-regulation and homeostasis of anionic phospholipids to maintain the membrane integrity. In addition, we identified an ethanolamine cytidyltransferase (TgECT), the rate-limiting enzyme of Kennedy pathway, which is localized in the cytosol. The enzyme is clearly essential for the lytic cycle as its genetic ablation was not feasible, and auxin-meditated conditional knockdown severely impaired the parasite growth in plaque assays. Similarly, lipidomic analysis of the mutant identified an important role in the biogenesis of selected species of PtdEtn, PtdSer and PtdThr. Moreover, we discovered that TgECT is required for the generation of ethanolamine-phosphory ceramide (EPC), a rare sphingolipid present only a limited number of organisms.

No description available.

Apicomplexa, einschließlich Plasmodium sp. (Erreger der Malaria) und Toxoplasma gondii (Erreger der Toxoplasmose), sind ein großes Phylum einzelliger, obligat intrazellulärer Parasiten, welche ein essenzielles, Plastiden ähnliches Organell, den sogenannten Apicoplast, beherbergen. Aufgrund der Beteiligung an mehreren essenziellen Stoffwechselprozessen stellt das Ferredoxin Redoxsystem innerhalb der Apicomplexa ein vielversprechendes potentielles Wirkstoffziel dar. Eine kürzlich veröffentlichte Arbeit zeigt, dass ptFd in T. gondii (TgFd) ein essenzielles Protein ist. In der vorliegenden Arbeit konnte durch eine gezielte Analyse von Metaboliten der Isoprenoid-Biosynthese gezeigt werden, dass T. gondii Fd eine essenzielle physiologische Funktion als Elektronendonor für die letzten beiden Enzyme dieses Stoffwechselweges hat. Weiterhin zeigen im Zusammenhang mit dieser Arbeit generierte Daten, das der Einfluss auf die Isoprenoid-Biosynthese einen zusätzlichen Effekt auf die Proteinprenylierung hat. Darüber hinaus zeigen die Ergebnisse dieser Arbeit, dass die Hemmung der Isoprenoid Biosynthese des Wirtes zum langsamen Einsetzen des Absterbens bei TgFd Knockdown-Parasiten beiträgt, was darauf hindeutet, dass der Mangel an Isoprenoid Vorstufen nach induziertem Fd Knockdown bis zu einem gewissen Grad durch vom Wirt stammende Isoprenoide kompensiert werden kann. Zusammenfassend tragen die Ergebnisse dieser Doktorarbeit zu einem besseren Verständnis der metabolischen Rolle von Ferredoxin in T. gondii und P. falciparum bei und zeigen dessen Bedeutung für den Parasitenstoffwechsel und verdeutlichen damit das Potenzial Ferredoxins als Wirkstoffziel gegen das große Phylum der Apicomplexa. / Apicomplexan parasites, including Plasmodium sp. (the causative agent of malaria) and Toxoplasma gondii (causing toxoplasmosis), are a large phylum of unicellular, obligate intracellular organisms. The plant type ferredoxin redox system in apicomplexan parasites is a promising drug target due to its potential involvement in several essential metabolic processes. Recently published work demonstrated that ptFd in T. gondii (TgFd) is an essential protein. A tetracycline-inducible knock-down (ikd) approach was used to replace the endogenous single copy of TgFd with a myc-tagged copy (TgFdmyc) by double cross-over homologous recombination, and severe growth inhibition of parasites was observed upon Fd depletion. Metabolomic analyses show a 30% decrease in C14:0 fatty acids and a significantly lower gliding motility (20%) in the TgFd ikd strain compared to the TgFd ikd complemented (TgFd cikd) strain. In this thesis, targeted metabolomic analysis of the isoprenoid biosynthesis metabolites demonstrates that T. gondii Fd has an essential physiological function as an electron donor for the last two enzymes of the pathway. Furthermore, results of this work show that inhibition of the host isoprenoid biosynthesis contributes to the slow onset of death in TgFd knockdown parasites, indicating that the lack of isoprenoid precursors after induced Fd knockdown can to some extent be compensated by host-derived isoprenoids. Together, the findings of this study contribute to a better understanding of the metabolic role of Fd in T. gondii and P. falciparum, supporting its importance for the parasite’s metabolism and underlining its potential as a drug target in apicomplexan parasites.

Das Überleben und die Vermehrung der parasitär lebenden Apicomplexa setzen eine effiziente Synthese von Phospholipiden während ihres gesamten Lebenszyklus voraus. In dieser Arbeit nutzten wir zunächst Eimeria falciformis um den Prozess der Lipid-Biogenese in Sporozoiten zu untersuchen. Durch Lipidomics-Analysen wurde das Auftreten von zwei exklusiven Lipiden, Phosphatidylthreonin (PtdThr) und Inositolphosphorylceramid. Der Parasit exprimiert fast das gesamte Lipid-Biogenese- Netzwerk aus eukaryotischen und prokaryotischen Enzymen. Toxoplasma gondii diente als genmanipulierbarer Ersatz für die Untersuchung der Eimeria-Enzyme, mit dem wir ein stark räumlich segmentiertes Netzwerk der Lipidsynthese im Apicoplast, ER, Golgi und Mitochondrium zeigen konnten. Ebenso legte die Komplementierung einer T. gondii-Mutante mit einer PtdThr-Synthase von E. falciformis eine konvergente Funktion von PtdThr für den lytischen Zyklus von Kokzidien-Parasiten nahe. Außerdem setzten wir T. gondii als etablierten Modelorganismus ein, um die De- novo-Synthese und die metabolische Rolle eines bedeutenden Lipidvorläufers, CDP- Diacylglycerin (CDP-DAG), zu untersuchen. Wir konnten zwei phylogenetisch divergente CDP-DAG-Synthase (CDS) Enzyme in T. gondii nachweisen. Das eukaryotisch-typische TgCDS1 und das prokaryotisch-typische TgCDS2 lokalisieren im ER bzw. im Apicoplast. Der konditionierte Knockdown von TgCDS1 bremst das Parasitenwachstum stark ab, was den fast vollständigen Verlust der Virulenz im Mausmodell hervorruft. Das restliche marginale Wachstum der TgCDS1 Mutante wird durch zusätzliche Deletion der TgCDS2 verhindert. Lipidomics-Analysen zeigten eine signifikante und spezifische Abnahme der Phosphatidylinositol (PtdIns)- und Phosphatidylglycerol (PtdGro)-Level bei Verlust der TgCDS1- bzw. TgCDS2-Gene. Zusammengenommen zeigt unsere Arbeit ein Phospholipid-Biogenese-Modell mit erstaunlicher Kooperation verschiedener Organellen und einem extensiven Lipidtransport im Parasiten. / The survival and proliferation of apicomplexan parasites oblige efficient synthesis of phospholipids throughout their life cycles. Here, we first deployed Eimeria falciformis to investigate the process of lipid biogenesis in sporozoites. Lipidomics analyses demonstrate the occurrence of two exclusive lipids phosphatidylthreonine (PtdThr) and inositol phosphorylceramide along with other prototypical lipids. The parasite expresses nearly the entire lipid biogenesis network, which is an evolutionary mosaic of eukaryotic- and prokaryotic-type enzymes. Using Toxoplasma gondii as a gene- tractable surrogate to examine the Eimeria enzymes, we show a highly compartmentalized network of lipid synthesis distributed primarily in the apicoplast, ER, Golgi and mitochondrion. Likewise, trans-species complementation of a T. gondii mutant with a PtdThr synthase from E. falciformis suggests a convergent function of PtdThr in promoting the lytic cycle in coccidian parasites. We also employed the well-established model parasite T. gondii to explore de novo synthesis and metabolic roles of one major lipid precursor CDP-diacylglycerol (CDP- DAG). We report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in T. gondii. Eukaryotic-type TgCDS1 and prokaryotic-type TgCDS2 reside in the ER and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuates parasite growth, which translates into a nearly complete loss of virulence in a mouse model. Residual growth of the TgCDS1 mutant is abolished by subsequent deletion of TgCDS2. Lipidomics analyses reveal significant and specific decline in phosphatidylinositol (PtdIns) and phosphatidylglycerol (PtdGro) upon loss of TgCDS1 and TgCDS2, respectively. Taken together, our work establishes a phospholipid biogenesis model involving significant inter-organelle cooperation and lipid trafficking in apicomplexan parasites.

Ma thèse s'inscrit dans l'étude des modifications du globule rouge humain induites par P. falciparum. Ces modifications qui représentent une remarquable adaptation du parasite à un environnement plus complexe qu'il n'y paraît au premier abord et expliquent sa persistance chez l'Homme sont détaillées dans une revue et un chapitre de livre dont je suis co-auteur. Mes travaux de recherche ont porté sur la caractérisation fonctionnelle des structures de Maurer, un compartiment membranaire exporté par le parasite dans le globule rouge parasitaire et directement lié à la physiopathologie du paludisme grave. J'ai contribué à la caractérisation fonctionnelle de nouvelles protéines de ces structures, codées par trois familles multigéniques sub-télomériques en cluster avec la famille Pfmc-2tm, et présentant de façon étonnante un fort degré de conservation (article 1). La diminution d'expression de ces gènes, obtenue par titration d'un facteur transcriptionnel, entraine un défaut de libération des mérozoïtes. Mon deuxième projet porte sur l'identification des modalités d'export de la protéine transmembranaire résidente des structures de Maurer PfSBP1. Mes travaux montrent que PfSBP1 est exportée sous forme soluble dans le cytoplasme érythrocytaire, en interaction avec le complexe chaperon parasitaire PfTCP1 (article 2). / Plasmodium falciparum causes the most severe forms of human malaria, a pathology associated with the erythrocytic asexual stages of the parasite. My work focused on the remodeling of the infected erythrocytes induced by P. falciparum and detailed in a review and a book chapter that I co-authored. These modifications illustrate a remarkable adaptation of P. falciparum resulting in its persistence in humans. My PhD thesis was dedicated to the functional characterization of Maurer's clefts, a membrane compartment transposed by the parasite in the cytoplasm of its host cell, and central to the export of virulence factors to the host cell surface. I have conducted two projects and contributed first to the functional characterization of novel exported protein encoded by three highly conserved multigene sub-telomeric families in cluster with the Pfmc-2tm family. Down regulation of these gene families by promoter titration impacted the release of infectious merozoites from the host cell (annex 1). My second project was dedicated to the identification of the modality of export of the resident and Maurer's clefts transmembrane protein PfSBP1. I have shown that PfSBP1 is exported as a soluble protein in the host cell cytoplasm in interaction with the parasite Thermosome complex protein 1 (PfTCP1) chaperone complex (annex 2).