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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 suppressive effect on E. cuniculi growth is impaired in GMS-IRG knock-out cells.(A) Wildtype (wt) or Irgm1/Irgm3 knock-out (KO) MEFs were induced with 200 U/ml IFNγ for 24 h and then infected with E. cuniculi spores for 24 h or left untreated. Cells were fixed and stained for meronts using 6G2 mAB (red) and host nuclei with DAPI (pseudocolored in cyan). Representative fluorescence microscopic images are shown. (B) Quantification of A, representative of two independent experiments. (C/D) Transformed wildtype or transformed IRG knock-out MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores or left untreated. Cells were harvested after 2 days (in D) and 5 days (in E) post-infection. Cell lysates were separated by SDS-PAGE and Western blots were cut into three regions and simultaneously probed for anti-meront mAB 6G2, anti-Calnexin pAB, which served as loading control and anti-Irgb6 (mAB B34) or anti-Irga6 (mAB 10E7 for Irgm1 KO and Irgm3 KO MEFs both at 5 d post infection; 165/3 pAS for Irgm1/Irgm3KO MEFs at 5 d post infection) as IFNγ-induction control. The black arrows highlight a 6G2-positive protein band indicating E. cuniculi growth despite presence of IFNγ, which is inhibited in wt cells (grew arrows). The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. The white asterisk marks unspecific bands. The four samples of one cell line per time point were analyzed together by one single SDS-PAGE and Western Blot, except for the Irgm1KO MEFs at 2 d post infection. The data represents at least three independent experiments.
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ppat-1004449-g004: IFNγ suppressive effect on E. cuniculi growth is impaired in GMS-IRG knock-out cells.(A) Wildtype (wt) or Irgm1/Irgm3 knock-out (KO) MEFs were induced with 200 U/ml IFNγ for 24 h and then infected with E. cuniculi spores for 24 h or left untreated. Cells were fixed and stained for meronts using 6G2 mAB (red) and host nuclei with DAPI (pseudocolored in cyan). Representative fluorescence microscopic images are shown. (B) Quantification of A, representative of two independent experiments. (C/D) Transformed wildtype or transformed IRG knock-out MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores or left untreated. Cells were harvested after 2 days (in D) and 5 days (in E) post-infection. Cell lysates were separated by SDS-PAGE and Western blots were cut into three regions and simultaneously probed for anti-meront mAB 6G2, anti-Calnexin pAB, which served as loading control and anti-Irgb6 (mAB B34) or anti-Irga6 (mAB 10E7 for Irgm1 KO and Irgm3 KO MEFs both at 5 d post infection; 165/3 pAS for Irgm1/Irgm3KO MEFs at 5 d post infection) as IFNγ-induction control. The black arrows highlight a 6G2-positive protein band indicating E. cuniculi growth despite presence of IFNγ, which is inhibited in wt cells (grew arrows). The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. The white asterisk marks unspecific bands. The four samples of one cell line per time point were analyzed together by one single SDS-PAGE and Western Blot, except for the Irgm1KO MEFs at 2 d post infection. The data represents at least three independent experiments.

Mentions: To assess the importance of IRG proteins in the IFNγ-dependent restriction of E. cuniculi, we investigated the development of the parasite in cells derived from IRG knock-out mice. First, we examined E. cuniculi infection in IFNγ-induced primary wildtype and Irgm1/Irgm3−/− MEF cells, which lack the two regulator GMS proteins, Irgm1 and Irgm3, and also express reduced levels of GKS proteins [40]. The number of meronts observed in Irgm1/Irgm3−/− MEF cells 24 h after infection was the same whether the cells were induced with IFNγ or not (Figure 4A, B). In contrast, and as observed in Figure 1, the number of meronts in IFNγ-induced wildtype cells was drastically reduced at 24 h after infection compared with uninduced controls (Figure 4A, B).


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 suppressive effect on E. cuniculi growth is impaired in GMS-IRG knock-out cells.(A) Wildtype (wt) or Irgm1/Irgm3 knock-out (KO) MEFs were induced with 200 U/ml IFNγ for 24 h and then infected with E. cuniculi spores for 24 h or left untreated. Cells were fixed and stained for meronts using 6G2 mAB (red) and host nuclei with DAPI (pseudocolored in cyan). Representative fluorescence microscopic images are shown. (B) Quantification of A, representative of two independent experiments. (C/D) Transformed wildtype or transformed IRG knock-out MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores or left untreated. Cells were harvested after 2 days (in D) and 5 days (in E) post-infection. Cell lysates were separated by SDS-PAGE and Western blots were cut into three regions and simultaneously probed for anti-meront mAB 6G2, anti-Calnexin pAB, which served as loading control and anti-Irgb6 (mAB B34) or anti-Irga6 (mAB 10E7 for Irgm1 KO and Irgm3 KO MEFs both at 5 d post infection; 165/3 pAS for Irgm1/Irgm3KO MEFs at 5 d post infection) as IFNγ-induction control. The black arrows highlight a 6G2-positive protein band indicating E. cuniculi growth despite presence of IFNγ, which is inhibited in wt cells (grew arrows). The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. The white asterisk marks unspecific bands. The four samples of one cell line per time point were analyzed together by one single SDS-PAGE and Western Blot, except for the Irgm1KO MEFs at 2 d post infection. The data represents at least three independent experiments.
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Related In: Results  -  Collection

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ppat-1004449-g004: IFNγ suppressive effect on E. cuniculi growth is impaired in GMS-IRG knock-out cells.(A) Wildtype (wt) or Irgm1/Irgm3 knock-out (KO) MEFs were induced with 200 U/ml IFNγ for 24 h and then infected with E. cuniculi spores for 24 h or left untreated. Cells were fixed and stained for meronts using 6G2 mAB (red) and host nuclei with DAPI (pseudocolored in cyan). Representative fluorescence microscopic images are shown. (B) Quantification of A, representative of two independent experiments. (C/D) Transformed wildtype or transformed IRG knock-out MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores or left untreated. Cells were harvested after 2 days (in D) and 5 days (in E) post-infection. Cell lysates were separated by SDS-PAGE and Western blots were cut into three regions and simultaneously probed for anti-meront mAB 6G2, anti-Calnexin pAB, which served as loading control and anti-Irgb6 (mAB B34) or anti-Irga6 (mAB 10E7 for Irgm1 KO and Irgm3 KO MEFs both at 5 d post infection; 165/3 pAS for Irgm1/Irgm3KO MEFs at 5 d post infection) as IFNγ-induction control. The black arrows highlight a 6G2-positive protein band indicating E. cuniculi growth despite presence of IFNγ, which is inhibited in wt cells (grew arrows). The asterisk marks an unknown E. cuniculi-derived protein that is detected by the Calnexin antibody. The white asterisk marks unspecific bands. The four samples of one cell line per time point were analyzed together by one single SDS-PAGE and Western Blot, except for the Irgm1KO MEFs at 2 d post infection. The data represents at least three independent experiments.
Mentions: To assess the importance of IRG proteins in the IFNγ-dependent restriction of E. cuniculi, we investigated the development of the parasite in cells derived from IRG knock-out mice. First, we examined E. cuniculi infection in IFNγ-induced primary wildtype and Irgm1/Irgm3−/− MEF cells, which lack the two regulator GMS proteins, Irgm1 and Irgm3, and also express reduced levels of GKS proteins [40]. The number of meronts observed in Irgm1/Irgm3−/− MEF cells 24 h after infection was the same whether the cells were induced with IFNγ or not (Figure 4A, B). In contrast, and as observed in Figure 1, the number of meronts in IFNγ-induced wildtype cells was drastically reduced at 24 h after infection compared with uninduced controls (Figure 4A, B).

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