Stable <i>T. gondii</i> parasite lines expressing transgenic OVA antigen.

<p>(A) OVA protein fused to endogenous targeting signals correctly traffics OVA antigen (green) to specific organelles in <i>T. gondii</i> tachyzoites, including: the cytoplasm (row 1, left), the parasitophorous vacuole (row 1, right), the inner membrane complex (row 2), the mitochondrion (row 3), and the parasite membrane (row 4). Permeabilization reveals intracellular, as well as membrane-bound, GPI-OVA (row 5). Co-localization markers (red) include: anti-IMC1 antibody, mitotracker, and anti-P30 antibody, as labeled. Protein expression in stable transgenic parasites (B) and culture supernatants (C) was analyzed by immunblotting, using antibodies directed against OVA and P30 (loading control).</p

Activation of OVA-specific B3Z T-cells by <i>T. gondii</i> expressing OVA antigen.

<p>Bone marrow derived macrophages (A) and dendritic cells (B) were stimulated with OT-I peptide or infected with <i>T. gondii</i> expressing OVA in various subcellular compartments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022936#pone-0022936-g001" target="_blank">Fig. 1</a>), and co-cultivated with B3Z T-cells in medium containing the ß-galactosidase substrate CPRG. Absorbance at 565 nm is represented relative to controls treated with media alone (average of three replicate assays). Results shown are representative of three independent experiments. Asterisks indicate <i>p</i><0.05 (*) or <i>p</i><0.005 (**), based on the students T-test statistic.</p

Proliferation of adoptively transferred OT-I T-cells after infection with OVA-expressing <i>T. gondii</i>.

<p>(A) CFSE levels of Thy1.2+ OT-I T-cells after murine infection with <i>T. gondii</i> expressing secreted or GPI-anchored OVA (top left) or intracellular OVA antigens (bottom left). Right-hand panels indicate the time course of OT-I activation in various organs (% CFSE-dull cells) following infection with P30-OVA or GPI-OVA. (B) CD62L (top) and CD44 (bottom) levels on Thy1.2+ OT-I T-cells after mice were infected with <i>T. gondii</i> expressing either secreted or GPI-anchored OVA (left, histograms; right, time course).</p

Transcriptional profiling of human Leishmania braziliensis skin lesions

<p>Normalized, batch corrected Log2 gene expression data and average Log2 FC for 20,942 genes across all 35 skin samples (10 normal, 8 early lesions, 17 late lesions) represented by one or more probesets from the Illumina HT-12v4 beadarray.</p> <p>Abbreviations: FC, fold-change.</p

Comparison of the L. braziliensis transcriptiome with published psoriasis datasets

<p>Comparison of transcriptional responses in L. braziliensis lesions with published psoriasis meta-analysis. Log2 FC in gene expression between skin lesion and normal control skin for 17,070 genes in common between the L. braziliensis data and published psoriasis ‘MAD3’ data. Rank order is shown for both Leishmania and psoriasis data in which genes were ranked from most highly upregulated in lesion (rank = 1) to most downregulated in lesion (rank = 17,070). Abbreviations: FC, fold change</p

Pathway analysis of differentially expressed genes in L. braziliensis lesions

<p>Pathway enrichment analysis. Excel spreadsheet showing pathways and their enrichment score from Fig. 2b found by GSEA to be enriched 16 fold (FDR ≤ 1%) in L. braziliensis lesions, relative to normal skin. Functionally related pathways manually grouped together for simplified representation in Fig. 2b are shown as a separate tab in the spreadsheet. Abbreviations: GSEA, gene set enrichment analysis; FDR, false discovery rate.</p

Differential induction of innate immune signaling by genetically related parasite species.

<p>a) Microarray-based expression profiling of human foreskin fibroblasts (HFF) infected with <i>Neospora caninum</i> (NcLiv isolate) or <i>Toxoplasma</i> (GT1, Prugniaud or VEG strains). Heat map shows hierarchical clustering analysis of 822 genes differentially regulated relative to uninfected cells by ≥2-fold (FDR≤5%), in any of these experiments. Each row in heatmap represents average of duplicate (NcLiv and GT1) or triplicate arrays (VEG, PRU, uninf). Color pattern on heatmaps represents column Z-score. b) A cluster of 66 genes (Fig. 1a, asterisk) induced by <i>Neospora</i> but not any strain of <i>Toxoplasma</i>, including several well-known type I interferon response genes (arrows). c) Gene Ontology (GO) enrichment analysis of 66 <i>Neospora</i>-induced genes. Bar graph shows fold enrichment for top five GO Biological Process terms enriched at <i>P</i>≤0.05 and represented by ≥5 genes. Number of genes and GO term name is shown at the right of each bar. d) Fluorescence micrographs of uninfected HFF cells, or cells infected with <i>Neospora</i> or <i>Toxoplasma</i>, then challenged with GFP-tagged vesicular stomatitis virus. Representative images are shown. Experiment was repeated three times with similar results.</p

The Type I interferon response to Neospora infection is dependent on <i>Tlr3</i> and <i>Trif.</i>

<p>QPCR analysis of <i>Mx1</i> expression in <i>Neospora</i> infected bone marrow macrophages derived from wild-type (WT), <i>Myd88^−/−</i>, <i>Myd88^−/−/Trif^−/−, Tlr2^−/−/Tlr4^−/−</i> and <i>Tlr3^−/−</i> mice. Error bars indicate standard deviations for three biological replicates; * = <i>P</i>≤0.01. Experiment was repeated three times with similar results.</p

Active invasion is not required for innate recognition of parasites.

<p>QPCR analysis of the expression of the antiviral gene <i>Mx1</i> following (a) infection of HFF cells or (b) treatment of cells with heat-killed strains of <i>Toxoplasma</i> (open bars) or <i>Neospora</i> (shaded bars). (c) confocal fluorescence microscopy of HFF cells treated with live (top row) or heat killed (bottom row) <i>Toxoplasma</i>-mCherry. Lysosomes are stained with LysoTracker dye. Representative images are shown. Error bars indicate standard deviations for three biological replicates; * = <i>P</i>≤0.01. Experiments were repeated three times with similar results.</p

<i>Neospora</i> RNA elicits a TLR3-dependent type I interferon response.

<p>a) QPCR analysis of <i>Irf7</i> gene expression in wild-type mouse bone marrow-derived macrophages pretreated for 1 hr with either DMSO (control) or 100 nM bafilomycin A1 prior to 24 hr infection with live (Nc) or heat-killed <i>Neospora</i> (HK Nc). b) <i>Irf7</i> expression in <i>Myd88^−/−</i> macrophages incubated with 1 µg/ml of either poly(I:C) or total RNA from HFF cells (host), or <i>Neospora</i> parasites, either alone (white) or in complex with DOTAP transfection reagent (striped), to enhance targeting of RNA to endosomes. c) <i>Irf7</i> expression in <i>Myd88^−/−</i> macrophages incubated with 1 µg/ml of either <i>Toxoplasma</i> or <i>Neospora</i> RNA with DOTAP in cells pretreated with either DMSO or a small molecule inhibitor of TLR3/dsRNA complex formation. Error bars indicate standard deviations for three biological replicates; * = <i>P</i>≤0.01. Experiments were repeated two or three times with similar results.</p