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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

Tryptophan supplementation cannot reverse the IFNγ-mediated E. cuniculi restriction.C57/BL/6 MEFs (A) or mouse enterocytic CMT-93 cells (B) were treated with IFNγ for 24 h or left uninduced. Indicated doses of L-Tryptophan (W) were added to the medium 30 minutes prior infection with E. cuniculi spores. Cell lysates were prepared 5 days post infection and separated by one SDS-PAGE. Western blots were cut into three regions probed simultaneously with for anti-meront mAB 6G2, anti-Calnexin pAB as loading control and anti-Irga6 (10E7 mAB) as IFNγ-induction control. The data are representative of three independent experiments.
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ppat-1004449-g006: Tryptophan supplementation cannot reverse the IFNγ-mediated E. cuniculi restriction.C57/BL/6 MEFs (A) or mouse enterocytic CMT-93 cells (B) were treated with IFNγ for 24 h or left uninduced. Indicated doses of L-Tryptophan (W) were added to the medium 30 minutes prior infection with E. cuniculi spores. Cell lysates were prepared 5 days post infection and separated by one SDS-PAGE. Western blots were cut into three regions probed simultaneously with for anti-meront mAB 6G2, anti-Calnexin pAB as loading control and anti-Irga6 (10E7 mAB) as IFNγ-induction control. The data are representative of three independent experiments.

Mentions: Restricting nutrient acquisition is a common defence mechanism against intracellular parasites. Deprivation of tryptophan by the IFN-inducible indoleamine 2,3-dioxygenase (IDO) is often claimed to be the main inhibitor of T. gondii replication in IFNγ-induced human fibroblasts [reviewed in [44], [45]], following reports that replication can be rescued by supplementation of the medium with tryptophan [46], [47]. IDO-mediated growth restriction of E. intestinalis has been proposed following observations in a mouse enterocytic cell line CMT-93 [33]. However, another study in activated mouse peritoneal macrophages showed that L-tryptophan supplementation failed to rescue the infection [48]. In view of these apparently inconsistent results, we analysed E. cuniculi growth in IFNγ-induced mouse cells following tryptophan supplementation (Figure 6). In wildtype MEF cells, as well as in CMT-93 cells, IFNγ-mediated growth restriction on E. cuniculi could not be reversed by supplementation with excess tryptophan, arguing strongly against mediation of the inhibition via IDO. Taken together with the complete loss of resistance caused by IRG protein deficiencies, we conclude that the IFNγ-mediated restriction of E. cuniculi in non-myeloid cells is mediated exclusively by the IRG system in mice.


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)

Tryptophan supplementation cannot reverse the IFNγ-mediated E. cuniculi restriction.C57/BL/6 MEFs (A) or mouse enterocytic CMT-93 cells (B) were treated with IFNγ for 24 h or left uninduced. Indicated doses of L-Tryptophan (W) were added to the medium 30 minutes prior infection with E. cuniculi spores. Cell lysates were prepared 5 days post infection and separated by one SDS-PAGE. Western blots were cut into three regions probed simultaneously with for anti-meront mAB 6G2, anti-Calnexin pAB as loading control and anti-Irga6 (10E7 mAB) as IFNγ-induction control. The data are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004449-g006: Tryptophan supplementation cannot reverse the IFNγ-mediated E. cuniculi restriction.C57/BL/6 MEFs (A) or mouse enterocytic CMT-93 cells (B) were treated with IFNγ for 24 h or left uninduced. Indicated doses of L-Tryptophan (W) were added to the medium 30 minutes prior infection with E. cuniculi spores. Cell lysates were prepared 5 days post infection and separated by one SDS-PAGE. Western blots were cut into three regions probed simultaneously with for anti-meront mAB 6G2, anti-Calnexin pAB as loading control and anti-Irga6 (10E7 mAB) as IFNγ-induction control. The data are representative of three independent experiments.
Mentions: Restricting nutrient acquisition is a common defence mechanism against intracellular parasites. Deprivation of tryptophan by the IFN-inducible indoleamine 2,3-dioxygenase (IDO) is often claimed to be the main inhibitor of T. gondii replication in IFNγ-induced human fibroblasts [reviewed in [44], [45]], following reports that replication can be rescued by supplementation of the medium with tryptophan [46], [47]. IDO-mediated growth restriction of E. intestinalis has been proposed following observations in a mouse enterocytic cell line CMT-93 [33]. However, another study in activated mouse peritoneal macrophages showed that L-tryptophan supplementation failed to rescue the infection [48]. In view of these apparently inconsistent results, we analysed E. cuniculi growth in IFNγ-induced mouse cells following tryptophan supplementation (Figure 6). In wildtype MEF cells, as well as in CMT-93 cells, IFNγ-mediated growth restriction on E. cuniculi could not be reversed by supplementation with excess tryptophan, arguing strongly against mediation of the inhibition via IDO. Taken together with the complete loss of resistance caused by IRG protein deficiencies, we conclude that the IFNγ-mediated restriction of E. cuniculi in non-myeloid cells is mediated exclusively by the IRG system in mice.

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