Cooperative binding of ApiAP2 transcription factors is crucial for the expression of virulence genes in Toxoplasma gondii

International audienceToxoplasma gondii virulence depends on the expression of factors packed into specific organelles such as rhoptry and microneme. Although virulence factor expression is tightly regulated, the molecular mechanisms controlling their regulation remain poorly understood. ApiAP2 are a family of conserved transcription factors (TFs) that play an important role in regulating gene expression in apicomplexan parasites. TgAP2XI-5 is able to bind to transcription-ally active promoters of genes expressed during the S/M phase of the cell cycle, such as virulence genes (rhoptries and micronemes genes). 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 demonstrated that the level of expression of number of virulence factors transcripts is affected by the disruption of TgAP2X-5 expression. While TgAP2X-5 disruption has mild effect on parasite 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 strain 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. Taken together, these data suggest a cooperative contribution of two ApiAP2 TF in the regulation of virulence genes in T. gondii

Nuclear Glycolytic Enzyme Enolase of Toxoplasma Gondii Functions as a Transcriptional Regulator

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii

Characterization of a nuclear pore protein sheds light on the roles and composition of the Toxoplasma gondii nuclear pore complex

International audienceThe nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes

A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

International audienceIn Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii

International Journal of Antimicrobial Agents A potent HDAC inhibitor blocks Toxoplasma gondii tachyzoite growth and profoundly disrupts parasite gene expression

International audienceToxoplasmosis is a major health issue worldwide especially for immune-deficient individuals and the offspring of newly infected mothers. It is caused by a unicellular intracellular parasite called Toxoplasma gondii. Although the drugs commonly used to treat toxoplasmosis are efficient, they present serious side effects and adverse events are common. Therefore, there is a need for the discovery of new compounds with potent anti-T. gondii activity. We have tested compounds designed to target enzymes that are involved in the epigenetic regulation of gene expression. Among the most active compounds, we identified an HDAC inhibitor that shows an IC 50 of around 30 nM with a selectivity index of more than 100. MC1742 is active at inhibiting the growth of the parasite in vitro but also at preventing the consequences of the acute disease in vivo. This compound induces hyper-acetylation of histones while acetylated tubulin level remains unchanged. After MC1742 treatment, the parasite expression profile is profoundly changed with the activation of genes preferentially expressed in the sexual stages that are normally repressed at the tachyzoite stage. These findings suggest that this compound disturbs the T. gondii gene expression program, inducing parasite death

Primary brain cell infection by Toxoplasma gondii reveals the extent and dynamics of parasite differentiation and its impact on neuron biology

International audienceToxoplasma gondii is a eukaryotic parasite that forms latent cysts in the brain of immunocompetent individuals. The latent parasite infection of the immune-privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons' long-term infection are unknown. It has long been known that T. gondii specifically differentiates into a latent form (bradyzoite) in neurons, but how the infected neuron responds to the infection remains to be elucidated. We have established a new in vitro model resulting in the production of mature bradyzoite cysts in brain cells. Using dual, host and parasite RNA-seq, we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal-specific pathways are strongly affected, with synapse signalling being particularly affected, especially glutamatergic synapse signalling. The establishment of this new in vitro model allows investigating both the dynamics of parasite differentiation and the specific response of neurons to long-term infection by this parasite

Nuclear Glycolytic Enzyme Enolase of <i>Toxoplasma gondii</i> Functions as a Transcriptional Regulator

<div><p>Apicomplexan parasites including <i>Toxoplasma gondii</i> have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that <i>T. gondii</i> enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin <i>in vivo</i>. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in <i>T. gondii</i>.</p></div

TgAP2IX-5 is a key transcriptional regulator of the asexual cell cycle division in Toxoplasma gondii

International audienceApicomplexan parasites have evolved efficient and distinctive strategies for intracellular replication where the timing of emergence of the daughter cells (budding) is a decisive element. However, the molecular mechanisms that provide the proper timing of parasite budding remain unknown. Using Toxoplasma gondii as a model Apicomplexan, we identified a master regulator that controls the timing of the budding process. We show that an ApiAP2 transcription factor, TgAP2IX-5, controls cell cycle events downstream of centrosome duplication. TgAP2IX-5 binds to the promoter of hundreds of genes and controls the activation of the budding-specific cell cycle expression program. TgAP2IX-5 regulates the expression of specific transcription factors that are necessary for the completion of the budding cycle. Moreover, TgAP2IX-5 acts as a limiting factor that ensures that asexual proliferation continues by promoting the inhibition of the differentiation pathway. Therefore, TgAP2IX-5 is a master regulator that controls both cell cycle and developmental pathways

Expression of ectopic TgENO2-HA and native TgENO2 proteins is predominantly nuclear and increases with intracellular replication of <i>T. gondii</i>.

<p>A) Kinetics of nuclear accumulation of ectopic TgENO2-HA protein in transgenic tachyzoites at different time points (0, 6, 12, 18, 24, 30, and 36 h post-infection) during the intracellular division cycle. Intracellular dividing transgenic tachyzoites were fixed and stained with rabbit polyclonal anti-HA and DAPI followed by confocal imaging. Scale bars, 5 µm. B) Kinetics of nuclear accumulation of native TgENO2 protein in wild type <i>T. gondii</i> tachyzoites at different time points (0, 6, 12, 18, 24, 30, and 36 h post-infection) during the intracellular division cycle. Intracellular dividing transgenic tachyzoites were fixed and stained with rabbit polyclonal anti-ENO2 antibodies and DAPI followed by confocal imaging. Scale bars, 5 µm. C) Quantification of cytoplasmic and nuclear levels of ectopic TgENO2-HA and native TgENO2 in intracellular dividing tachyzoites of <i>T. gondii</i>. Experiments were repeated three times (n = 3, P<0.001). Quantifications were performed on at least 8–10 independent intracellular vacuoles using ImageJ software and bioinformatics tools as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105820#s2" target="_blank">Materials and Methods</a>.</p

Targeted deletion of TgENO1 and complementation of the knockout demonstrate that nuclear enolase modulates gene transcript levels.

<p>A) Total RNA extracted from intracellular tachyzoites of Pru<i>Δku80ΔTgeno1</i> mutants or parental Pru<i>Δku80</i> grown in normal culture conditions was reverse transcribed and used for quantitative RT-PCR. The eight enolase target genes selected by ChIP-Seq and beta tubulin, a housekeeping gene, were analyzed. B) Total RNA was extracted from intracellular tachyzoites of the Pru<i>Δku80ΔTgeno1</i> mutant or parental Pru<i>Δku80</i> after bradyzoite interconversion <i>in vitro</i> using alkaline (pH 8) stress culture conditions. After reverse transcription, the cDNA was subjected to quantitative RT-PCR analysis of the eight enolase target genes selected by ChIP-Seq and beta tubulin. C) Total RNA extracted from intracellular tachyzoites of Pru<i>Δku80ΔTgeno1</i> mutants complemented with TgENO1 plasmid or parental Pru<i>Δku80</i> parasites grown in normal culture conditions was reverse transcribed and used for quantitative RT-PCR analysis of the eight enolase target genes and beta tubulin. D) Total RNA was extracted from intracellular tachyzoites from Pru<i>Δku80ΔTgeno1</i> mutant complemented with TgENO1 plasmid or parental Pru<i>Δku80</i> parasites after bradyzoite interconversion <i>in vitro</i> using alkaline (pH 8) stress culture conditions. After reverse transcription, the cDNA was subjected to quantitative RT-PCR analysis of the eight enolase target genes selected by ChIP-Seq and beta tubulin. These experiments were performed at least three times with identical results (n = 3, P<0.0001).</p