O parasita Plasmodium falciparum causa a forma mais grave da malária humana.Para evadir a resposta imune do hospedeiro, as formas assexuadas do parasita podem usar variação antigênica ou podem se diferenciar em formas sexuais como estratégia para sobreviver e garantir a sua transmissão para o mosquito.A base molecular desses processos ainda é pouco compreendida. Por manipulação genética, nos identificamos a participação de um fator de transcrição da família ApiAP2 ( PF3D7_1143100), no controle da transcrição de genes variantes e no desenvolvimento em formas sexuais na fase intraeritrocítica. Demonstramos ainda que um outro membro desta família, PF3D7_1466400, não é essencial no ciclo assexual de P. falciparum, já que seu silenciamento não afeto o normal desenvolvimento do parasita. / The parasite Plasmodium falciparum causes the most severe form of human malaria. To evade the host immune response, asexual parasite forms can employ antigenic variation or differentiation to gametocytes as a means to survive and secure their transmission to the mosquito. The molecular basis behind these processes is still poorly understood. By genetic manipulation, we indentified the participation of a ApiAP2 transcription factor, PF3D7_1143100, in the control of variant gene transcription as well as in the switching from asexual to sexual development in the intraerythrocytic stage. We also demonstrate that the ApiAP2 transcription factor PF3D7_1466400 is not essential in the asexual stage of P. falciparum, since its knockdown did not affect the normal development of the parasite.

O parasita Plasmodium falciparum causa a forma mais grave da malária humana.Para evadir a resposta imune do hospedeiro, as formas assexuadas do parasita podem usar variação antigênica ou podem se diferenciar em formas sexuais como estratégia para sobreviver e garantir a sua transmissão para o mosquito.A base molecular desses processos ainda é pouco compreendida. Por manipulação genética, nos identificamos a participação de um fator de transcrição da família ApiAP2 ( PF3D7_1143100), no controle da transcrição de genes variantes e no desenvolvimento em formas sexuais na fase intraeritrocítica. Demonstramos ainda que um outro membro desta família, PF3D7_1466400, não é essencial no ciclo assexual de P. falciparum, já que seu silenciamento não afeto o normal desenvolvimento do parasita. / The parasite Plasmodium falciparum causes the most severe form of human malaria. To evade the host immune response, asexual parasite forms can employ antigenic variation or differentiation to gametocytes as a means to survive and secure their transmission to the mosquito. The molecular basis behind these processes is still poorly understood. By genetic manipulation, we indentified the participation of a ApiAP2 transcription factor, PF3D7_1143100, in the control of variant gene transcription as well as in the switching from asexual to sexual development in the intraerythrocytic stage. We also demonstrate that the ApiAP2 transcription factor PF3D7_1466400 is not essential in the asexual stage of P. falciparum, since its knockdown did not affect the normal development of the parasite.

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.

Toxoplasma gondii est un eucaryote unicellulaire appartenant au phylum des Apicomplexes. Ce parasite intracellulaire obligatoire est la cause d’une pathologie sévère pour les fœtus des femmes enceintes primo-infectées ainsi que pour les personnes immunodéprimées. Son cycle de vie est complexe et comporte plusieurs étapes de prolifération et différenciation. Le stade tachyzoïte est responsable de la phase aigüe de la maladie chez les humains. Le tachyzoïte exprime des facteurs d'invasion et de virulence cruciaux pour sa survie et le détournement de la cellule hôte. L'expression de ces facteurs de virulence est hautement régulée pour permettre leur adressage correct dans des organites spécifiques appelés rhoptries et micronèmes. Cependant, peu d'informations sont connues sur les acteurs contrôlant l'expression de ces gènes de virulence. Les ApiAP2 appartiennent à une famille conservée de facteur de transcription (FT) et jouent un rôle important dans la régulation de la transcription des gènes chez les parasites apicomplexes. Des études menées au laboratoire ont révélés la capacité du FT TgAP2XI-5 à se fixer sur des promoteurs de gènes transcriptionnellement actifs durant les phases S/M du cycle cellulaire. Ce moment correspond au pic d’expression les gènes de rhoptrie et de micronème. Cependant, l'expression de TgAP2XI-5 est constitutive durant le cycle cellulaire chez le tachyzoïte. Dans le but de comprendre comment sa fonction dépendante du cycle cellulaire est régulée, nous avons identifié des potentiels partenaires d'interactions dont TgAP2X-5, un autre FT ApiAP2 dont l’expression est régulée durant les phases S/M du cycle. L'utilisation d'un système d'expression inductible ainsi que des expériences de séquençage des ARN nous ont permis de démontrer que la perte d'expression de TgAP2X-5 induit une diminution du nombre de transcrits codant pour les protéines de rhoptrie et de micronème. Alors que la perte d'expression de TgAP2X-5 n'influence pas de manière significative l'invasion et la croissance du parasite, nous observons une perte totale de virulence de la souche parasitaire in vivo. Pour mieux comprendre les mécanismes moléculaires mis en jeu, nous nous sommes demandés si la fixation de TgAP2XI-5 sur ces promoteurs cible est conservée en absence de TgAP2X-5. Par des expériences de ChIP-chip, nous avons montrés que l'absence de TgAP2X-5 conduit à une incapacité de fixation de TgAP2XI-5 sur des promoteurs de gènes de rhoptrie. L'ajout d'une copie du gène TgAP2X-5 sous son propre promoteur dans la souche mutante est capable de restaurer l'expression des protéines de rhoptrie préalablement affectée. Toutes ces expériences montrent pour la première fois une coopération des FT APiAP2 dans la régulation des gènes chez les Apicomplexes. / Toxoplasma gondii is a unicellular eukaryote belonging to the Apicomplexa phylum. It is an obligate intracellular parasite of critical importance to primarily infected pregnant women and immunosuppressed patient. Its life cycle is complex, with multiple proliferation and differentiation steps, of which tachyzoite proliferation is the most relevant to pathogenesis in humans. Tachyzoites express invasion and virulence factors that are crucial for their survival and the manipulation of host cell functions. Expression of these virulence factors is tightly regulated permitting their correct targeting in specific organelles named rhoptry and microneme. However, little is known about the factors controlling the expression of genes encoding the virulence factors. ApiAP2 are a family of conserved transcription factors (TF) that play an important role in regulating gene expression in apicomplexan parasites. Previous studies had revealed the ability of one of these TFs, TgAP2XI-5, to bind to transcriptionally active promoters of genes expressed during the S/M phase such as rhoptry and micronemes genes. However, expression of TgAP2XI-5 is constitutive during the tachyzoite cell cycle. To better understand how its function is regulated, we identified proteins interacting with TgAP2XI-5 including a cell cycle regulated ApiAP2 TF, TgAP2X-5. Using an inducible knock-down strategy and RNA-Seq, we demonstrate that the level of expression of number of rhoptry and microneme transcripts is affected by the disruption of TgAP2X-5 expression. While TgAP2X-5 disruption has mild effect on parasite growth and invasion, it leads to the strain avirulence in mice. To better understand the molecular mechanisms at stake, we investigated the binding of TgAP2XI-5 at promoters in the TgAP2X-5 mutant in a genome-wide assay. We show that disruption of TgAP2X-5 expression leads to defects in TgAP2XI-5 binding to multiple rhoptry gene promoters. Complementation of the TgAP2X-5 mutant restores the expression of rhoptry proteins previously affected. This is the first evidence of a cooperative action of ApiAP2 TF in Apicomplexa.