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

Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite Toxoplasma gondii

The protozoan parasite Toxoplasma gondii differentially expresses two distinct enolase isoenzymes known as ENO1 and ENO2, respectively. To understand differential gene expression during tachyzoite to bradyzoite conversion, we have characterized the two T.gondii enolase promoters. No homology could be found between these sequences and no TATA or CCAAT boxes were evident. The differential activation of the ENO1 and ENO2 promoters during tachyzoite to bradyzoite differentiation was investigated by deletion analysis of 5′-flanking regions fused to the chloramphenicol acetyltransferase reporter followed by transient transfection. Our data indicate that in proliferating tachyzoites, the repression of ENO1 involves a negative distal regulatory region (nucleotides −1245 to −625) in the promoter whereas a proximal regulatory region in the ENO2 promoter directs expression at a low level. In contrast, the promoter activity of ENO1 is highly induced following the conversion of tachyzoites into resting bradyzoites. The ENO2 promoter analysis in bradyzoites showed that there are two upstream repression sites (nucleotides −1929 to −1067 and −456 to −222). Furthermore, electrophoresis mobility shift assays demonstrated the presence of DNA-binding proteins in tachyzoite and bradyzoite nuclear lysates that bound to stress response elements (STRE), heat shock-like elements (HSE) and other cis-regulatory elements in the upstream regulatory regions of ENO1 and ENO2. Mutation of the consensus AGGGG sequence, completely abolished protein binding to an oligonucleotide containing this element. This study defines the first characterization of cis-regulatory elements and putative transcription factors involved in gene regulation of the important pathogen T.gondii

Étude de la O-N-acétylglucosaminylation chez le parasite Toxoplasma gondii et de son rôle dans la localisation nucléo-cytopasmique des énolases

L'inter-conversion du tachyzoïte virulent en bradyzoïte quiescent est au centre de la pathogenèse et de la pérennité du parasite protozoaire Toxoplasma gondii. Les bases moléculaires régissant cette différenciation parasitaire n'ont pas encore été complètement élucide es à ce jour. L'inter-conversion est marquée par l'expression spécifique de stade d'un grand nombre de protéines, conséquente à une régulation fine des gènes du parasite. Notre laboratoire a notamment mis en évidence l'existence de deux protéines homologues de l' énolase, ENO 1 exprimée chez le bradyzoïte, et ENO2 exprimée chez le tachyzoïte. Ces deux enzymes glycolytiques, ordinairement cytoplasmiques, sont localisées dans le noyau des parasites pendant la réplication active et au cours du processus d'inter-conversion. L'obtention de tachyzoïtes transgéniques sur-exprimant ENOl ou ENO2 en fusion avec l'épitope HA, a permis de confirmer la localisation nucléaire des deux protéines et de mettre en évidence l'induction de l'expression de gènes spécifiques du stade bradyzoite, suggérant un rôle de régulateur génique de ces enzymes glycolytiques. Une cinétique in vitro de l'adressage nucléaire d'ENO2 dans le tachyzoïte a révélé que la localisation nucléaire de cette enzyme était corrélée aux stades précoces de la réplication parasitaire. L'étude du processus autorisant la localisation nucléo-cytoplasmique des deux énolases a permis de démontrer que ENO1 et probablement ENO2 portent un ou plusieurs résidus O-GlcNAc, modification posttraductionnelle agissant de concert avec la phosphorylation et permettant la localisation nucléocytoplasmique. Le gène codant pour l'enzyme permettant de greffer ces résidus O-GlcNAc sur les protéines, la O-GlcNAc transférase ou OGT, a été identifié par bio-informatique et partiellement cloné. L'analyse par Blast de l'OGT putative ainsi qu'une étude phylogénétique ont mis en évidence la forte homologie de cette protéine avec les OGTs végétales, les protéines SPINDLY, rappelant le lien entre les Apicomplexa et les micro-organismes photosynthétiques. Ce travail démontre pour la première fois l'existence de la O- N-acétylglucosaminylation et de l'OGT chez Toxoplasma gondii.LILLE1-BU (590092102) / SudocSudocFranceF

A Toxoplasma gondii Leucine-Rich Repeat Protein Binds Phosphatase Type 1 Protein and Negatively Regulates Its Activity▿

We have characterized the Toxoplasma gondii protein phosphatase type 1 (TgPP1) and a potential regulatory binding protein belonging to the leucine-rich repeat protein family, designated TgLRR1. TgLRR1 is capable of binding to TgPP1 to inhibit its activity and to override a G2/M cell cycle checkpoint in Xenopus oocytes. In the parasite, TgLRR1 mRNA and protein are both highly expressed in the rapidly replicating and virulent tachyzoites, while only low levels are detected in the slowly dividing and quiescent bradyzoites. TgPP1 mRNA and protein levels are equally abundant in tachyzoites and bradyzoites. Affinity pull down and immunoprecipitation experiments reveal that the TgLRR1-TgPP1 interaction takes place in the nuclear subcompartment of tachyzoites. These results are consistent with those of localization studies using both indirect immunofluorescence with specific polyclonal antibody and transient transfection of T. gondii vector expressing TgLRR1 and TgPP1. The inability to obtain stable transgenic tachyzoites suggested that overexpression of TgLRR1 and TgPP1 may impair the parasite's growth. Together with the activation of Xenopus oocyte meiosis reinitiation, these data indicate that TgLRR1 protein could play a role in the regulation of the T. gondii cell cycle through the modulation of phosphatase activity

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

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

Role of the TTTTCT motif in specific TgENO2-DNA interactions and promoter activity.

<p>A) The TTTTCT motif was identified in the putative gene promoters targeted by nuclear TgENO2 using ChIP-Seq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105820#pone.0105820.s008" target="_blank">Table S4</a>) and the MEME bioinformatics tool (a motif-based sequence analysis tool). B) Nucleotide sequences of probes corresponding to the TTTTCT motif in the promoter of TgMag1 gene, the TATA box from human c-Myc gene, and a non-relevant motif used as a negative control. C) Expression and purification of recombinant TgENO2 fused to His-Tag. D) Electrophoretic band shift assays using recombinant TgENO2 incubated with or without the probes described in panel A. The unlabeled competitor was present at 100-fold excess. E) The GCTAGC motif is required for efficient transcription of the TgMag1 gene. The putative promoter of TgMag1, corresponding to a 787-bp region upstream of the start codon, was subjected to site-directed mutagenesis resulting in sequential disruption of the single TTTCT motif within the TTTTTCTTCTC motif of TgMag1 to <i><u>ATCGA</u></i>TCTC (<i>Δ</i>₁<i>Tg</i>Mag1) and then to <i><u>ATCGAGCGC</u></i> (<i>Δ</i>₂<i>Tg</i>MAg2). These two mutant promoters and the wild-type promoter were cloned upstream of a reporter luciferase construct and assayed for their ability to drive transcription. The transcriptional potential of mutated promoters was measured as firefly luciferase activity normalized to activity of a vector encoding <i>Renilla</i> luciferase. These experiments have been performed three times (n = 3, p<0.001).</p

Validation of native TgENO2 and transgenic TgENO2-HA protein bound to several putative gene promoters.

<p>A) Quantitative real-time PCR analysis of chromatin immunoprecipitates from three independent experiments (n = 3, P<0.0001) demonstrates specific binding of nuclear TgENO2 <i>in vivo</i> to eight selected genes identified by ChIP-Seq (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105820#pone.0105820.s008" target="_blank">Table S4</a>). A gene encoding a hypothetical protein that was absent from the gene hits (TgME49_ 0022080) was used as a negative control. B) Quantitative real-time PCR analysis of chromatin immunoprecipitates from three independent experiments (n = 3, P<0.0001) demonstrates specific binding of nuclear transgenic TgENO2-HA <i>in vivo</i> to eight selected genes identified by ChIP-Seq (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105820#pone.0105820.s008" target="_blank">Table S4</a>). The TgME49_ 0022080 gene was used as a negative control.</p

Targeted deletion of TgENO1 reduces cyst burden in the brain of chronically infected mice.

<p>A) The total number of cysts per brain of mice infected with 5×10² tachyzoites from the Pru<i>Δku80ΔTgeno1</i> mutant or parental Pru<i>Δku80</i> was counted after staining with FITC-labeled <i>dolichol biflorus</i> lectin. A group of nine mice was used for each experiment, and the experiment was repeated twice with similar results (n = 2, P<0.001). Cyst burden (total number of cysts per brain) of the Pru<i>Δku80ΔTgeno1</i> mutant was significantly lower than that of the parental Pru<i>Δku80</i> strain. B) Western blots of total SDS-extracted proteins from knockout Pru<i>Δku80ΔTgeno1</i> mutants (lane 1) and parental Pru<i>Δku80</i> tachyzoites (lane 2). Left panel was probed with the polyclonal anti-ENO2 antibodies while the right panel was stained with the monoclonal anti-actin antibodies. C) Western blots of mutants and wild type parasites. Lane 1, total SDS-protein extracts from wild type 76K tachyzoites. Lane 2, total SDS-extracted proteins from transgenic E1-5 tachyzoites. Lane 3, total SDS-extracted proteins from transgenic E2-4. Lane 4, total SDS-extracted proteins from transgenic E2-10. Lane 5, total SDS-extracted proteins from parental Pru<i>Δku80</i> tachyzoites. Lane 6, total SDS-extracted proteins from knock-out Pru<i>Δku80ΔTgeno1</i> mutants. Blots were stained with polyclonal antibodies specific to LDH1, LDH2, G6PI and monoclonal antibodies specific to actin. The numbers on the left indicate molecular markers in kilodaltons.</p