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Identification of the microsporidian Encephalitozoon cuniculi as a new target of the IFNγ-inducible IRG resistance system.

Ferreira-da-Silva Mda F, da Fonseca Ferreira-da-Silva M, Springer-Frauenhoff HM, Bohne W, Howard JC - PLoS Pathog. (2014)

Bottom Line: We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle.The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function.The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT
The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

No MeSH data available.


Related in: MedlinePlus

IFNγ restricts E. cuniculi growth in mouse embryonic fibroblasts.(A) Mouse embryonic fibroblasts (MEFs) from C57BL/6 mice were induced with IFNγ for 24 h or left uninduced before infection with E. cuniculi spores. Cells were fixed at the indicated time points and the number of meronts (stained with anti-meront mAb 6G2) per 500 host nuclei (stained with DAPI) was counted. The inhibition in the IFNγ-treated sample compared to the uninduced control sample is presented as mean +/− standard deviation (SD) of 3–7 replicates per time point from at least 2 individual experiments. Significant differences (of 0.5 h, 1 h and 2–3 h compared to 24–26 h) were calculated with a two tailed T-test. (B) MEFs were induced with IFNγ or left uninduced, infected with E. cuniculi spores for 24 h and stained as in A. Single meronts and meronts that divided once (double meront) were counted per 500 host nuclei and shown as percent of total vacuoles of uninduced controls. Numbers indicate the counted number of single or double meronts per 500 host cells. Data from three independent experiments (Exp. 1–3) is presented. (C) MEFs were stimulated with IFNγ and/or infected with E. cuniculi spores for 2 or 5 days or left untreated. Cell lysates were separated by SDS-PAGE and Western Blots were cut into three regions to probe for anti-meront mAB 6G2 as well as anti-spore wall protein 1 pAS SWP1. Calnexin staining served as loading control and Irgb6 staining (mAB B34) to proof IFNγ-induction. The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. These Western Blots emerged from one single SDS-PAGE, the 45–70 kDa region was first probed with mouse mAB B34, stripped, and then probed for anti-SWP1 rabbit pAS. Experiments for both time points were performed at least three times.
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ppat-1004449-g001: IFNγ restricts E. cuniculi growth in mouse embryonic fibroblasts.(A) Mouse embryonic fibroblasts (MEFs) from C57BL/6 mice were induced with IFNγ for 24 h or left uninduced before infection with E. cuniculi spores. Cells were fixed at the indicated time points and the number of meronts (stained with anti-meront mAb 6G2) per 500 host nuclei (stained with DAPI) was counted. The inhibition in the IFNγ-treated sample compared to the uninduced control sample is presented as mean +/− standard deviation (SD) of 3–7 replicates per time point from at least 2 individual experiments. Significant differences (of 0.5 h, 1 h and 2–3 h compared to 24–26 h) were calculated with a two tailed T-test. (B) MEFs were induced with IFNγ or left uninduced, infected with E. cuniculi spores for 24 h and stained as in A. Single meronts and meronts that divided once (double meront) were counted per 500 host nuclei and shown as percent of total vacuoles of uninduced controls. Numbers indicate the counted number of single or double meronts per 500 host cells. Data from three independent experiments (Exp. 1–3) is presented. (C) MEFs were stimulated with IFNγ and/or infected with E. cuniculi spores for 2 or 5 days or left untreated. Cell lysates were separated by SDS-PAGE and Western Blots were cut into three regions to probe for anti-meront mAB 6G2 as well as anti-spore wall protein 1 pAS SWP1. Calnexin staining served as loading control and Irgb6 staining (mAB B34) to proof IFNγ-induction. The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. These Western Blots emerged from one single SDS-PAGE, the 45–70 kDa region was first probed with mouse mAB B34, stripped, and then probed for anti-SWP1 rabbit pAS. Experiments for both time points were performed at least three times.

Mentions: A time series of 0.5 to 24 hours post infection showed continuous IFNγ-dependent loss of E. cuniculi meronts (determined by counting of 6G2-positive meronts per host nuclei). Meront numbers at the earliest time point measured (0.5 hours) were equivalent in IFNγ-treated and untreated cells showing that E. cuniculi invasion into the host cells was not significantly affected by prior IFNγ induction (Figure 1A). Next, we quantified not only single meronts, but also meronts that had replicated by binary fission (double meronts) and compared uninduced to IFNγ-induced MEF cells 24 hours post infection. Of the few surviving meronts at 24 h, very few had successfully divided in IFNγ-induced cells (Figure 1B).


Identification of the microsporidian Encephalitozoon cuniculi as a new target of the IFNγ-inducible IRG resistance system.

Ferreira-da-Silva Mda F, da Fonseca Ferreira-da-Silva M, Springer-Frauenhoff HM, Bohne W, Howard JC - PLoS Pathog. (2014)

IFNγ restricts E. cuniculi growth in mouse embryonic fibroblasts.(A) Mouse embryonic fibroblasts (MEFs) from C57BL/6 mice were induced with IFNγ for 24 h or left uninduced before infection with E. cuniculi spores. Cells were fixed at the indicated time points and the number of meronts (stained with anti-meront mAb 6G2) per 500 host nuclei (stained with DAPI) was counted. The inhibition in the IFNγ-treated sample compared to the uninduced control sample is presented as mean +/− standard deviation (SD) of 3–7 replicates per time point from at least 2 individual experiments. Significant differences (of 0.5 h, 1 h and 2–3 h compared to 24–26 h) were calculated with a two tailed T-test. (B) MEFs were induced with IFNγ or left uninduced, infected with E. cuniculi spores for 24 h and stained as in A. Single meronts and meronts that divided once (double meront) were counted per 500 host nuclei and shown as percent of total vacuoles of uninduced controls. Numbers indicate the counted number of single or double meronts per 500 host cells. Data from three independent experiments (Exp. 1–3) is presented. (C) MEFs were stimulated with IFNγ and/or infected with E. cuniculi spores for 2 or 5 days or left untreated. Cell lysates were separated by SDS-PAGE and Western Blots were cut into three regions to probe for anti-meront mAB 6G2 as well as anti-spore wall protein 1 pAS SWP1. Calnexin staining served as loading control and Irgb6 staining (mAB B34) to proof IFNγ-induction. The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. These Western Blots emerged from one single SDS-PAGE, the 45–70 kDa region was first probed with mouse mAB B34, stripped, and then probed for anti-SWP1 rabbit pAS. Experiments for both time points were performed at least three times.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214799&req=5

ppat-1004449-g001: IFNγ restricts E. cuniculi growth in mouse embryonic fibroblasts.(A) Mouse embryonic fibroblasts (MEFs) from C57BL/6 mice were induced with IFNγ for 24 h or left uninduced before infection with E. cuniculi spores. Cells were fixed at the indicated time points and the number of meronts (stained with anti-meront mAb 6G2) per 500 host nuclei (stained with DAPI) was counted. The inhibition in the IFNγ-treated sample compared to the uninduced control sample is presented as mean +/− standard deviation (SD) of 3–7 replicates per time point from at least 2 individual experiments. Significant differences (of 0.5 h, 1 h and 2–3 h compared to 24–26 h) were calculated with a two tailed T-test. (B) MEFs were induced with IFNγ or left uninduced, infected with E. cuniculi spores for 24 h and stained as in A. Single meronts and meronts that divided once (double meront) were counted per 500 host nuclei and shown as percent of total vacuoles of uninduced controls. Numbers indicate the counted number of single or double meronts per 500 host cells. Data from three independent experiments (Exp. 1–3) is presented. (C) MEFs were stimulated with IFNγ and/or infected with E. cuniculi spores for 2 or 5 days or left untreated. Cell lysates were separated by SDS-PAGE and Western Blots were cut into three regions to probe for anti-meront mAB 6G2 as well as anti-spore wall protein 1 pAS SWP1. Calnexin staining served as loading control and Irgb6 staining (mAB B34) to proof IFNγ-induction. The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. These Western Blots emerged from one single SDS-PAGE, the 45–70 kDa region was first probed with mouse mAB B34, stripped, and then probed for anti-SWP1 rabbit pAS. Experiments for both time points were performed at least three times.
Mentions: A time series of 0.5 to 24 hours post infection showed continuous IFNγ-dependent loss of E. cuniculi meronts (determined by counting of 6G2-positive meronts per host nuclei). Meront numbers at the earliest time point measured (0.5 hours) were equivalent in IFNγ-treated and untreated cells showing that E. cuniculi invasion into the host cells was not significantly affected by prior IFNγ induction (Figure 1A). Next, we quantified not only single meronts, but also meronts that had replicated by binary fission (double meronts) and compared uninduced to IFNγ-induced MEF cells 24 hours post infection. Of the few surviving meronts at 24 h, very few had successfully divided in IFNγ-induced cells (Figure 1B).

Bottom Line: We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle.The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function.The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Cologne, Germany.

ABSTRACT
The IRG system of IFNγ-inducible GTPases constitutes a powerful resistance mechanism in mice against Toxoplasma gondii and two Chlamydia strains but not against many other bacteria and protozoa. Why only T. gondii and Chlamydia? We hypothesized that unusual features of the entry mechanisms and intracellular replicative niches of these two organisms, neither of which resembles a phagosome, might hint at a common principle. We examined another unicellular parasitic organism of mammals, member of an early-diverging group of Fungi, that bypasses the phagocytic mechanism when it enters the host cell: the microsporidian Encephalitozoon cuniculi. Consistent with the known susceptibility of IFNγ-deficient mice to E. cuniculi infection, we found that IFNγ treatment suppresses meront development and spore formation in mouse fibroblasts in vitro, and that this effect is mediated by IRG proteins. The process resembles that previously described in T. gondii and Chlamydia resistance. Effector (GKS subfamily) IRG proteins accumulate at the parasitophorous vacuole of E. cuniculi and the meronts are eliminated. The suppression of E. cuniculi growth by IFNγ is completely reversed in cells lacking regulatory (GMS subfamily) IRG proteins, cells that effectively lack all IRG function. In addition IFNγ-induced cells infected with E. cuniculi die by necrosis as previously shown for IFNγ-induced cells resisting T. gondii infection. Thus the IRG resistance system provides cell-autonomous immunity to specific parasites from three kingdoms of life: protozoa, bacteria and fungi. The phylogenetic divergence of the three organisms whose vacuoles are now known to be involved in IRG-mediated immunity and the non-phagosomal character of the vacuoles themselves strongly suggests that the IRG system is triggered not by the presence of specific parasite components but rather by absence of specific host components on the vacuolar membrane.

No MeSH data available.


Related in: MedlinePlus