Toxoplasma gondii is a single-celled parasite that persists in its host as a transmissible tissue cyst. How the parasite converts from its replicative form to the bradyzoites housed in tissue cysts is not well understood, but the process clearly involves changes in gene expression. Here we report that parasites lacking a cell cycle-regulated transcription factor called AP2IX-4 display reduced frequencies of tissue cyst formation in culture and in a mouse model of infection. Parasites missing AP2IX-4 lose the ability to regulate bradyzoite genes during tissue cyst development. Expressed in developing bradyzoites still undergoing division, AP2IX-4 may serve as a useful marker in the study of transitional forms of the parasite.

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host.

SUMMARY Parasitic infections remain one of the most pressing global health concerns of our day, affecting billions of people and producing unsustainable economic burdens. The rise of drug-resistant parasites has created an urgent need to study their biology in hopes of uncovering new potential drug targets. It has been established that disrupting gene expression by interfering with lysine acetylation is detrimental to survival of apicomplexan (Toxoplasma gondii and Plasmodium spp.) and kinetoplastid (Leishmania spp. and Trypanosoma spp.) parasites. As “readers” of lysine acetylation, bromodomain proteins have emerged as key gene expression regulators and a promising new class of drug target. Here we review recent studies that demonstrate the essential roles played by bromodomain-containing proteins in parasite viability, invasion, and stage switching and present work showing the efficacy of bromodomain inhibitors as novel antiparasitic agents. In addition, we performed a phylogenetic analysis of bromodomain proteins in representative pathogens, some of which possess unique features that may be specific to parasite processes and useful in future drug development.

Bradyzoite differentiation is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important Toxoplasma transcriptional repressor mechanism controlling bradyzoite differentiation that operates exclusively in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the increased expression of bradyzoite mRNAs in replicating tachyzoites, and in two different genetic lineages we confirmed the misexpression of tissue cyst wall components (e.g. BPK1, MCP4, CST1) and the bradyzoite surface antigen SRS9 in the tachyzoite stage. In the murine animal model, the loss of AP2IV-4 had profound biological consequences. Type II prugniaud strain parasites lacking AP2IV-4 were unable to form tissue cysts in brain tissue and the absence of this factor also recruited a potent immune response characterized by increases inflammatory monocytes, IFN-γ and higher numbers of both CD8+ and CD4+ T-cells. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to establish a chronic infection in the immune-competent host.

Overexpression of the GLI1 gene has frequently been found in various cancer types, particularly in brain tumors, in which aberrant GLI1 induction promotes cancer cell growth. Therefore, identifying the molecular players controlling GLI1 expression is of clinical importance. Previously, we reported that AMPK directly phosphorylated and destabilized GLI1, resulting in the suppression of the Hedgehog signaling pathway. The current study not only demonstrates that AMPK inhibits GLI1 nuclear localization, but further reveals that β-TrCP plays an essential role in AMPK-induced GLI1 degradation. We found that activation of AMPK promotes the interaction between β-TrCP and GLI1, and induces β-TrCP-mediated GLI1-ubiquitination and degradation. Inhibiting AMPK activity results in the dissociation of the β-TrCP and GLI1 interaction, and diminishes β-TrCP-mediated-GLI1 ubiquitination and degradation. On GLI1, substitution of AMPK phosphorylation sites to aspartic acid (GLI13E) results in stronger binding affinity of GLI1 with β-TrCP, accompanied by enhanced GLI1 ubiquitination and later degradation. In contrast, the GLI1 alanine mutant (GLI13A) shows weaker binding with β-TrCP, which is accompanied by reduced β-TrCP-mediated ubiquitination and degradation. Together, these results demonstrate that AMPK regulates GLI1 interaction with β-TrCP by phosphorylating GLI1 and thus both post-translational modifications by AMPK and β-TrCP ultimately impact GLI1 degradation.