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.

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.