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

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

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

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

Complementation of Pru<i>Δku80ΔTgeno1</i> mutants by ectopic expression of TgENO1-HA and growth under bradyzoite culture conditions.

<p>A) Western blot analysis of Pru<i>Δku80ΔTgeno1</i> mutants using total SDS-extracted antigens from <i>in vitro</i>-induced intracellular bradyzoites and probed with monoclonal antibodies specific to <i>T. gondii</i> actin (lane 1), bradyzoite-specific major surface protein 1 (anti-P36, lane 2), the influenza hemagglutinin (HA)-tag (anti-HA, lane 3) and TgENO1 (lane 4). B) Western blot analysis of complemented Pru<i>Δku80ΔTgeno1</i> mutants using total SDS-extracted antigens from <i>in vitro</i>-induced intracellular bradyzoites and probed with monoclonal antibodies specific to <i>T. gondii</i> actin (lane 1), bradyzoite-specific major surface protein (anti-P36, lane 2), the influenza hemagglutinin (HA)-tag (anti-HA, lane 3) and TgENO1 (lane 4). C) Confocal microscopy of <i>in vitro</i>-induced intracellular bradyzoites of Pru<i>Δku80ΔTgeno1</i> mutants that were grown in confluent HFF cells and stained with polyclonal anti-HA antibodies. The panels show nuclei stained with DAPI (upper left panel), IFA (upper right panel), phase contrast (lower left panel), and merged images (lower right panel). Scale bars are 5 µm. D) Confocal microscopy of <i>in vitro</i>-induced intracellular bradyzoites of complemented Pru<i>Δku80ΔTgeno1</i> mutants that were grown in confluent HFF cells and stained with anti-HA antibodies (upper panels show phase contrast, DAPI, IFA, and merged images). Complemented Pru<i>Δku80ΔTgeno1</i> mutants were grown in confluent HFF cells before double staining with FITC-labeled <i>dolichol biflorus</i> lectin (cyst wall marker, green) and polyclonal anti-HA antibodies (red). The nuclei were stained by DAPI (left middle panel, blue). IFA reveals staining of the cyst wall (right middle panel, green) and ectopic TgENO1-HA protein expressed in the complemented Pru<i>Δku80ΔTgeno1</i> mutants was stained by anti-HA antibodies (lower left panel, red). Merged images are shown in the lower right panel. Scale bars are 5 µm.</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

Identification of transcriptional regulatory elements in the and genes by transient CAT assays

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Transfections of tachyzoites were performed with () and () promoter–reporter constructs that are schematically depicted on the left of the panels. The transfected tachyzoites were also subjected to stress thereby converting to bradyzoites (see Materials and Methods). () Activities of the and promoters during tachyzoite to bradyzoite conversion. The plasmids E2F or E1F contain the 3′-UTR of gene while E2F10 and E1F9 contain the 3′-UTR of and , respectively. The CAT-constructs depicted on the left-hand side (E2F versus E2F10 and E1F versus E1F9) were transfected into tachyzoites and these organisms were divided equally into two flasks of human foreskin fibroblasts. After 6 h of invasion, one flask was subjected to experimental stress conditions to induce bradyzoite conversion and the second flask was kept in tachyzoite growth conditions (see Materials and Methods). Levels of CAT signal in tachyzoites and bradyzoites were determined after removing the promoterless CAT vector activities and adjustments for β-galactosidase activity used as internal control. The data are averages of four independent experiments. Error bars represent the mean and SD values of four independent experiments

The proximal regulatory elements of promoter is a target for DNA-binding proteins

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () A schematic representation showing the 5′-flanking region of that was used to generate DNA fragments for proteins binding in EMSA. The two fragments E2/2 and E2/3 binding specifically to proteins in EMSA using the tachyzoite nuclear extracts are indicated. () The nucleotide sequence of the contiguous E2/2 and E2/3 fragments is depicted with the oligonucleotides further designed for protein binding in EMSA. () Band shift assay with oligonucleotides from E2/2 and E2/3. Oligonucleotides 3a, 3b and 3c: lane 1, free probe; lane 2, no competitor; lane 3, specific competitor (50-fold or 100-fold excess); and oligonucleotides 2a and 2b: lane 1, free probe; lane 2, no competitor; lane 3, specific competitor (150-fold excess). The arrowheads show the DNA–protein complexes

Increased DNA–protein interactions with nuclear extract from stress-induced bradyzoites

<p><b>Copyright information:</b></p><p>Taken from "Transcriptional regulation of two stage-specifically expressed genes in the protozoan parasite "</p><p>Nucleic Acids Research 2005;33(5):1722-1736.</p><p>Published online 22 Mar 2005</p><p>PMCID:PMC1903550.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Electrophoretic band shift assays showing DNA–protein complexes with promoter DNA oligonucleotides containing STRE motifs (5b1 and 5b2) or HSE-like motifs (7b+7c) and nuclear extract from non-stressed tachyzoites (lanes 1–3), nuclear extract from stress-induced bradyzoites (lanes 4–6) or nuclear extract from heat shock treated tachyzoites (oligo 7b+7c, lanes 7–9). Equal amount of nuclear extracts have been used. Lane 1, free probe; lane 2, probe with tachyzoite nuclear extract and no competitor; lane 3, probe with tachyzoite nuclear extract and specific competitor (200-fold excess of the homologous cold fragment); lane 4, free probe; lane 5, probe with stress-induced bradyzoite nuclear extract and specific competitor (200-fold excess); lane 6, probe with stress-induced bradyzoite nuclear extract and specific competitor (200-fold excess); lane 7, free probe; lane 8, probe with nuclear extract from tachyzoite heat shock treated and no competitor; and lane 9, probe with tachyzoite nuclear extract and specific competitor (200-fold excess). Arrowheads show the DNA–protein complexes