Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen

Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation

Cyst Detection in Toxoplasma gondii Infected Mice and Rats Brain

Bio-protocol: http://bio-protocol.org/e1439[Abstract] Toxoplasmosis caused by the intracellular parasite Toxoplasma gondii, is characterized by a life-long chronic infection. The parasite is an efficient neurotropic infectious agent that establishes its “safe” life by forming intracellular cysts in chronically infected animals and humans. This protocol describes the specific recipes and method to stain brain cysts from infected mice and rats for further quantification using epifluorescence microscopy. This method provides the possibility to scan the entire brain and thus to numerate all cysts

Refractoriness to toxoplasmosis results from rapid and atypical death of infected macrophage and is associated to a highly conserved polymorphic haplotypic block

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A highly conserved toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage.

International audienceNatural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1β. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-β secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance

Reprogramming reactive glia into interneurons reduces chronic seizure activity in a mouse model of mesial temporal lobe epilepsy

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Allelic variations in <i>Toxo1</i> correlating with toxoplasmosis resistance.

<p>Sequencing of <i>Toxo1</i> (between 57.26 and 58.15 Mb) revealed 373 SNPs (red bars) conserved in coding- and non-coding sequences of all resistant strains and missing in susceptible strains. The diagram illustrates the distribution of these SNPs along the sequence of <i>Toxo1</i> (indicated by the double arrow in the lower part). In the upper part, the 29 genes are represented by black bold lines. The four genes named <i>Inca1</i>, <i>Kif1C</i>, <i>Nlrp1a</i> and <i>Nlrp1b</i> display missense mutations.</p

Intravacuolar Toxoplasma parasites are killed within resistant <i>Toxo1</i>-LEW peritoneal macrophages.

<p>(<b>A, B</b>) Macrophages settled on coverslips were infected with RH-YFP₂ (green) parasites for 2 h or 8 h, fixed and stained with anti-GRA5 (red) and Hoechst 33258 (blue); <b>A</b>: representative IF images (Bars: 5 µm); <b>B</b>: Quantification of GRA5-positive vacuoles containing YFP-positive parasites (mean ± SD of three experiments; _**: <i>p</i><0.01). (<b>C, D</b>) Macrophages on coverslips were infected with RH parasites for 2 h or 8 h prior to fixation and stained with anti-SAG1 (green), anti-GRA3 (red) and Hoechst 33258 (blue). <b>C</b>: Representative IF images (Bars: 5 µm); <b>D</b>: Quantification of GRA3-positive vacuoles containing SAG1-labelled parasites (mean ± SD of two experiments; _*: <i>p</i><0.05). (<b>E, F</b>) Macrophages on coverslips were infected with RH parasites for 2 h or 8 h prior to fixation and stained with anti-GRA5 (green), anti-Tg small ubiquitin-like modifier (SUMO, red) and Hoechst 33258 (blue). <b>E</b>: representative IF images (Bars: 5 µm); <b>F</b>: Quantification of GRA5-positive vacuoles containing SUMO-positive parasites (mean ± SD of three experiments; _**: <i>p</i><0.01). (<b>G</b>) Rat macrophages from congenic lines were infected by the RH-YFP₂ parasite strain. The YFP-positive infected cells were monitored by cytometry. Parasite death was evaluated through the ratio of YFP-positive infected cells between 6 h and 1 h post-infection. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004005#s2" target="_blank">Results</a> obtained for the LEW (n = 8) and congenic lines BN.LEWc10-Ce (n = 3), -Cf (n = 3), -Cg (n = 2), -Cga (n = 4), -Ch (n = 4), -Ci (n = 2), LEW.BNc10-F (n = 5) were normalized to the BN (n = 5) and represented by column showing means +/− SD; _*: <i>p</i><0.05; _**: <i>p</i><0.01. Genotypes at the markers of the <i>Toxo1</i> locus in the used BN.LEWc10 congenic lines are specified (N: homozygous for the BN genome; L: homozygous for the lew genome) and the grey zone indicates the <i>Toxo1</i> locus narrowed down to 0.89 Mb (boundary markers: D10GF49 and D10GF55; Physical distances are indicated between markers in kilobases).</p

The caspase-1 pathway controls the cell death induction of both host macrophages and intracellular parasite but not parasite proliferation.

<p>(<b>A, B, C</b>) Caspase-1 inhibitor YVAD and pan-caspase inhibitor Z-VAD (Calbiochem) were applied at respectively 50 µM and 100 µM to LEW and LEW.BNc10-F peritoneal macrophages infected 6 h with RH-YFP parasites. (<b>A</b>) Cell death was monitored by PI uptake. Histograms represent the percentage of PI positive dying cells (results indicate the difference between infected and uninfected cells). Data are means +/− SD from three independent experiments; _*: <i>p</i><0.05. (<b>B</b>) The YFP-positive infected macrophages were monitored by fluorescent microscopy. Parasite viability was evaluated through the percentage of YFP-positive infected cells 6 h post-infection. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004005#s2" target="_blank">Results</a> were normalized according to the percentage of YFP-positive infected cells obtained in untreated LEW.BNc10-F macrophages. Columns represent mean +/− SD of three different experiments; _*: <i>p</i><0.05; _**: <i>p</i><0.01. (<b>C</b>) Intracellular growth of <i>T. gondii</i> was measured by monitoring [³H] uracil incorporation into <i>T. gondii</i>. For each treatment, results were normalized according to the values obtained in LEW.BNc10-F macrophages. Columns represent mean +/− SD of three independent experiments; _*: <i>p</i><0.05; _**: <i>p</i><0.01.</p

The <i>Toxo1</i>-mediated death of <i>T. gondii</i>-infected macrophages is associated to ROS production and caspase-1 activation.

<p>(<b>A</b>) LEW and LEW.BNc10-F were infected 15 min or 4 h with <i>T. gondii</i>, then dihydro-rhodamine was added, and 15 min later the percentage of cells producing ROS was determined by flow cytometry. Histograms represent the percentage of cells producing ROS in uninfected and infected macrophages. Columns and bars show mean ± SD of three independent experiments; _*, <i>p</i><0.05. (<b>B</b>) Representative IF images of LEW and LEW.BNc10-F macrophages infected 4 h with RH parasites and labelled with FLICA assay (green) prior to fixation and Hoescht staining (blue). (<b>C, D</b>) LEW and LEW.BNc10-F macrophages were infected for 1 h and 4 h with RH parasites and then labelled with FLICA assay (green) and PI (red) prior to fixation and Hoescht staining (blue). (<b>C</b>) Histograms represent analysis of FLICA positive cells from two independent experiments. Data are means +/− SD. (<b>D</b>) Histograms represent analysis of FLICA and PI positive cells from two independent experiments. Data are means +/− SD. (<b>E</b>) <i>T. gondii</i> triggers caspase-1 dependent processing of IL-1β in resistant LEW but not in permissive Lew.BNc10-F macrophages. IL-1β precursor and mature IL-1β were analyzed by western blotting of cell lysates (Lys.) and culture supernatants (Sup.) from LEW and Lew.BNc10-F macrophages uninfected (NI) or infected for 1 h or 4 h with or without 50 µM of capase-1 inhibitor YVAD (Calbiochem). Immunoblot was normalized using equivalent number of cells for each condition and validated with anti-actin immunoblot.</p

Genetic dissection narrows down <i>Toxo1</i> to 0.89 megabase.

<p>(<b>A</b>) Genetic dissection based on <i>in vitro</i> phenotypes. [³H] uracil incorporation into <i>T. gondii</i> RNA as a read-out of parasite proliferation within macrophages <i>in vitro</i>; results are normalized according to the values obtained in BN macrophages. Columns and bars show mean ± SD, n = 4 except LEW (n = 8) and BN.LEWc10-Cf (n = 3); _*: <i>p</i><0.05; _**: <i>p</i><0.01 as compared to BN). (<b>B–D</b>) Genetic dissection based on <i>in vivo</i> phenotypes. (<b>B</b>) Anti-<i>T. gondii</i> IgG response and cyst number were analyzed in BN, LEW and six congenic BN.LEWc10 lines. Anti-<i>T. gondii</i> IgG Ab response was analyzed by ELISA at day 30 post infection. (<b>C</b>) Number of brain cysts was determined by epifluorescence at day 60. (<b>C–D</b>) Columns and bars show mean ± s.e.m, n = 5 except for BN.LEWc10-Ce and Cga (n = 4); _*:<i>p</i><0.05; _**:<i>p</i><0.01 (as compared to BN). (<b>D</b>) Genotypes at the markers of the <i>Toxo1</i> locus in the used BN.LEWc10 congenic lines. The grey zone indicates the <i>Toxo1</i> locus narrowed down to 0.89 Mb (boundary markers: D10GF49 and D10GF55; physical distances are indicated between markers in kilobases).</p