Ultrastructure of <i>Toxoplasma gondii</i>.

<p>(<b>A</b>) Intracellular <i>T. gondii</i> tachyzoite showing the MICs (Mn), ROPs (Rh), micropore (Mp), Golgi (G), nucleus (N), endoplasmic reticulum (ER), and dense granules (DG). (<b>B</b>) A schematic picture of <i>T. gondii</i> entering into a nucleated mammalian host cell. The apical exocytosis of MICs deploys onto the parasite surface MIC proteins required for parasite motility and the formation of moving junction. ROP secretion provides the ROP proteins that are involved in host cell invasion and modulation of immune responses. The constitutive secretion of dense granules (DG) is involved in the modification of the parasitophorous vacuole (PV). (<b>C</b>) Higher magnification of the single Golgi apparatus of <i>T. gondii</i>. (<b>D</b>) Higher magnification of the <i>T. gondii</i> ER. Scale bars, 1 µm.</p

Comparative bioinformatics analysis of genes coding components of vesicle-mediated trafficking and endosomal sorting in apicomplexan parasites, <i>Saccharomyces cerevisiae</i>, and <i>Homo sapiens</i>.

<p>Most of these genes and their corresponding accession numbers were collected from Eupathdb.org (for apicomplexan parasites) and uniprot.org (yeast and human cells). The data from apicomplexan parasites <i>Toxoplasma gondii</i> (<i>T. gondii</i>), <i>Plasmodium falciparum</i> (<i>P. falciparum</i>), <i>Theileria parva</i> (<i>T. parva</i>), and <i>Cryptosporidium parvum</i> (<i>C. parvum</i>) were compared with human (<i>H. sapiens</i>) and the yeast <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>). AP, adaptor protein; GGAs, Golgi-localized, γ-ear–containing, ADP-ribosylation factor binding protein; COPI, Coatomer complex I (retrograde transport from trans-Golgi apparatus to cis-Golgi and endoplasmic reticulum); COPII, Coatomer complex II (anterograde transport from ER to the cis-Golgi); ESCRT, Endosomal Sorting Complex Required for Transport.</p

A model for TgSORTLR functions in protein sorting and the biogenesis of apical secretory organelles.

<p>We propose that TgSORTLR has a distinct role as a type I transmembrane cargo-protein receptor for ROPs and MICs of apicomplexan parasites. We observed TgSORTLR-positive structures that could be transport vesicles destined for the endolysosomal system or they might be integral to the endolysosomal system, i.e., early (EE) and late (LE) endosomes. The model further proposes that the cytoplasmic tail of TgSORTLR binds to AP-1, Sec23/24, clathrin, clathrin-associated adaptor protein, and VPS9, and this defines it as a key receptor involved in the anterograde transport of cargo ROP and MIC proteins. The binding of TgSORTLR to the retromer VPS26/VPS35 also indicates that this receptor is also involved in the retrograde transport of components. <i>T. gondii</i> lysosome-like, acidic vacuolar compartment (VAC), also termed the Plant-Like Vacuole (PLV), contains cathepsin proteases implicated in the proteolytic maturation of proproteins targeted to MICs. Proteolytic maturation likely occurs in the LE where conditions are thought to be more conducive for limited proteolysis.</p

TgSORTLR co-localizes with TgVsp26 and Tgμ1-adpatin.

<p>(<b>A</b>) Confocal images of tachyzoites expressing endogenously tagged TgVps26-HA (green) that co-localizes with TgSORTLR (red). (<b>B</b>) Confocal images of tachyzoites expressing endogenously tagged Tgμ1adaptin-HA (red) and TgSORTLR (green). White circles indicate the zoomed areas showing co-distribution between TgSORTLR and Tgμ1adaptin-HA or TgVPS26-HA in the Golgi and post-Golgi. Scale bars, 5 µm.</p

Effect of Dex and implication of the GR in sPLA2-IIA promoter activity.

<p>A/ 158N cells were transiently transfected with 0.2 µg of sPLA2-IIA(1 kb) and 0,1 µg of pRSV-βGal plasmids in the presence of 0.2 µg of mock vector (NT) or siRNA directed against the GR (siGR). Eighteen hours after transfection, cells were incubated with 25-OH (10⁻⁵ M) and/or Dex (10⁻⁶ M) for 24 h, and then luciferase and β-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity; they represent the mean +/− SEM of 10 independent experiments performed in duplicate. In all experiments *p<0.05, **p<0.01, ***p<0.001 by using Bonferroni's test after ANOVA. B/ Test of the efficacy of the siRNA: Total RNA from 158N cells transfected with either non targeting siRNA or siRNA against GR was prepared. Real time RT-PCR was performed. 26S RNA was detected by specific primers and used to normalize GR expression levels.</p

Effect of dexamethasone on the expression of LXRβ and PXR.

<p>158N cells were incubated with Dex (1 µM) during 24 h. Total RNA was extracted and real-time PCR experiments were performed using primers recognizing specifically LXRβ or PXR. Results are expressed as the ratio of LXR or PXR expression over 26S. 100% is the level in control cells. They represent the mean +/− SEM of three independent experiments. *p<0.05 when compared to control by using Student's t test.</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

PGC-1α implication in sPLA2-IIA promoter regulation.

<p>158N cells were transiently transfected with 0.2 µg of sPLA2-IIA(1 kb), 0.1 µg of pRSV-βGal plasmids and with 0.1 µg of PGC-1α expression vector or mock vector, as indicated. Eighteen hours after transfection, cells were incubated with 25-OH (10⁻⁵ M) and/or Dex (10⁻⁶ M) for 24 h, and then luciferase and β-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity; they represent the mean +/− SEM of at least four independent experiments performed in duplicate. **p<0.01, when comparing between 25-OH and Dex+25-OH using Bonferroni's test after ANOVA.</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

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