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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.

Cavailles P, Flori P, Papapietro O, Bisanz C, Lagrange D, Pilloux L, Massera C, Cristinelli S, Jublot D, Bastien O, Loeuillet C, Aldebert D, Touquet B, Fournié GJ, Cesbron-Delauw MF - PLoS Pathog. (2014)

Bottom Line: 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.The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: UMR 5163, Centre National de la Recherche Scientifique (CNRS), Grenoble, France; Université Grenoble 1, Grenoble, France.

ABSTRACT
Natural 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.

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Genetic dissection narrows down Toxo1 to 0.89 megabase.(A) Genetic dissection based on in vitro phenotypes. [3H] uracil incorporation into T. gondii RNA as a read-out of parasite proliferation within macrophages in vitro; 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); *: p<0.05; **: p<0.01 as compared to BN). (B–D) Genetic dissection based on in vivo phenotypes. (B) Anti-T. gondii IgG response and cyst number were analyzed in BN, LEW and six congenic BN.LEWc10 lines. Anti-T. gondii IgG Ab response was analyzed by ELISA at day 30 post infection. (C) Number of brain cysts was determined by epifluorescence at day 60. (C–D) Columns and bars show mean ± s.e.m, n = 5 except for BN.LEWc10-Ce and Cga (n = 4); *:p<0.05; **:p<0.01 (as compared to BN). (D) Genotypes at the markers of the Toxo1 locus in the used BN.LEWc10 congenic lines. The grey zone indicates the Toxo1 locus narrowed down to 0.89 Mb (boundary markers: D10GF49 and D10GF55; physical distances are indicated between markers in kilobases).
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ppat-1004005-g001: Genetic dissection narrows down Toxo1 to 0.89 megabase.(A) Genetic dissection based on in vitro phenotypes. [3H] uracil incorporation into T. gondii RNA as a read-out of parasite proliferation within macrophages in vitro; 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); *: p<0.05; **: p<0.01 as compared to BN). (B–D) Genetic dissection based on in vivo phenotypes. (B) Anti-T. gondii IgG response and cyst number were analyzed in BN, LEW and six congenic BN.LEWc10 lines. Anti-T. gondii IgG Ab response was analyzed by ELISA at day 30 post infection. (C) Number of brain cysts was determined by epifluorescence at day 60. (C–D) Columns and bars show mean ± s.e.m, n = 5 except for BN.LEWc10-Ce and Cga (n = 4); *:p<0.05; **:p<0.01 (as compared to BN). (D) Genotypes at the markers of the Toxo1 locus in the used BN.LEWc10 congenic lines. The grey zone indicates the Toxo1 locus narrowed down to 0.89 Mb (boundary markers: D10GF49 and D10GF55; physical distances are indicated between markers in kilobases).

Mentions: We previously demonstrated that the Toxo1 7.6 Mb interval fully controls the outcome of T. gondii infection independently of the genetic background. The refractoriness to infection conferred by the LEW origin of Toxo1 is characterized by the early elimination of the pathogen resulting in a barely detectable specific immune response and in the absence of brain cysts [10]. In vitro, this Toxo1-LEW mediated refractoriness is associated with the control of parasite proliferation within macrophages [10]. To refine the localisation of the gene(s) that control(s) these in vivo and in vitro phenotypes, we generated a unique panel of congenic sub-lines. Results from the genetic dissection are shown on Figure 1. The parasites were found able to proliferate within the macrophages from the congenic BN.LEWc10-Ce, -Cf, -Cga, -Ci and LEW.BNc10-F sub-lines but not within the macrophages from the congenic BN.LEWc10-Cg and -Ch sub-lines (Figure 1A). Thus within the 7.6 Mb of the Toxo1 locus a 891 kb region controls the in vitro proliferation of parasites within macrophages. We further investigated refractoriness or susceptibility to T. gondii infection in vivo in rats from the seven congenic sub-lines used for these in vitro studies as well as in rats from the BN and LEW parental strains. The control of refractoriness to T. gondii defined by both the absence or low specific antibody response (Figure 1B) and the absence of cyst burden in the brain (Figure 1C), was directed by the same 891 kb region (Figure 1D). Thus, the interval located between the D10GF49 (57.26 Mb) and D10GF55 (58.15 Mb) microsatellite markers contains the gene or the set of genes that controls the toxoplasmosis outcome.


A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage.

Cavailles P, Flori P, Papapietro O, Bisanz C, Lagrange D, Pilloux L, Massera C, Cristinelli S, Jublot D, Bastien O, Loeuillet C, Aldebert D, Touquet B, Fournié GJ, Cesbron-Delauw MF - PLoS Pathog. (2014)

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

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ppat-1004005-g001: Genetic dissection narrows down Toxo1 to 0.89 megabase.(A) Genetic dissection based on in vitro phenotypes. [3H] uracil incorporation into T. gondii RNA as a read-out of parasite proliferation within macrophages in vitro; 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); *: p<0.05; **: p<0.01 as compared to BN). (B–D) Genetic dissection based on in vivo phenotypes. (B) Anti-T. gondii IgG response and cyst number were analyzed in BN, LEW and six congenic BN.LEWc10 lines. Anti-T. gondii IgG Ab response was analyzed by ELISA at day 30 post infection. (C) Number of brain cysts was determined by epifluorescence at day 60. (C–D) Columns and bars show mean ± s.e.m, n = 5 except for BN.LEWc10-Ce and Cga (n = 4); *:p<0.05; **:p<0.01 (as compared to BN). (D) Genotypes at the markers of the Toxo1 locus in the used BN.LEWc10 congenic lines. The grey zone indicates the Toxo1 locus narrowed down to 0.89 Mb (boundary markers: D10GF49 and D10GF55; physical distances are indicated between markers in kilobases).
Mentions: We previously demonstrated that the Toxo1 7.6 Mb interval fully controls the outcome of T. gondii infection independently of the genetic background. The refractoriness to infection conferred by the LEW origin of Toxo1 is characterized by the early elimination of the pathogen resulting in a barely detectable specific immune response and in the absence of brain cysts [10]. In vitro, this Toxo1-LEW mediated refractoriness is associated with the control of parasite proliferation within macrophages [10]. To refine the localisation of the gene(s) that control(s) these in vivo and in vitro phenotypes, we generated a unique panel of congenic sub-lines. Results from the genetic dissection are shown on Figure 1. The parasites were found able to proliferate within the macrophages from the congenic BN.LEWc10-Ce, -Cf, -Cga, -Ci and LEW.BNc10-F sub-lines but not within the macrophages from the congenic BN.LEWc10-Cg and -Ch sub-lines (Figure 1A). Thus within the 7.6 Mb of the Toxo1 locus a 891 kb region controls the in vitro proliferation of parasites within macrophages. We further investigated refractoriness or susceptibility to T. gondii infection in vivo in rats from the seven congenic sub-lines used for these in vitro studies as well as in rats from the BN and LEW parental strains. The control of refractoriness to T. gondii defined by both the absence or low specific antibody response (Figure 1B) and the absence of cyst burden in the brain (Figure 1C), was directed by the same 891 kb region (Figure 1D). Thus, the interval located between the D10GF49 (57.26 Mb) and D10GF55 (58.15 Mb) microsatellite markers contains the gene or the set of genes that controls the toxoplasmosis outcome.

Bottom Line: 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.The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: UMR 5163, Centre National de la Recherche Scientifique (CNRS), Grenoble, France; Université Grenoble 1, Grenoble, France.

ABSTRACT
Natural 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.

Show MeSH
Related in: MedlinePlus