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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

IRG proteins accumulate at the E. cuniculi parasitophorous vacuolar membrane.(A–F) MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 24 h. Fixed cells were stained for anti-meront mAB 6G2 as well as for endogenous Irga6 (165/3 pAS and 10D7 mAB), Irgb6 (A20 pAB), Irgd (2078 pAS) and Irgm2 (H53 pAS). Nuclei were labeled with DAPI. Representative microscopic images of IRG-positive E. cuniculi PVMs are presented. Yellow arrows point at the IRG-loaded PVM which is magnified at the end of each panel (zoom in the following order: upper left: merged image, upper right: phase contrast, lower left: anti-meront, lower right anti-IRG); white arrow: unloaded meront, every scale bar is 10 µm. (G) Quantification of Irga6 and Irgb6 loading onto the E. cuniculi PVM at different time points post infection. 100 vacuoles were evaluated per sample, a black dot indicates that sample was not counted, three independent experiments are shown.
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ppat-1004449-g002: IRG proteins accumulate at the E. cuniculi parasitophorous vacuolar membrane.(A–F) MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 24 h. Fixed cells were stained for anti-meront mAB 6G2 as well as for endogenous Irga6 (165/3 pAS and 10D7 mAB), Irgb6 (A20 pAB), Irgd (2078 pAS) and Irgm2 (H53 pAS). Nuclei were labeled with DAPI. Representative microscopic images of IRG-positive E. cuniculi PVMs are presented. Yellow arrows point at the IRG-loaded PVM which is magnified at the end of each panel (zoom in the following order: upper left: merged image, upper right: phase contrast, lower left: anti-meront, lower right anti-IRG); white arrow: unloaded meront, every scale bar is 10 µm. (G) Quantification of Irga6 and Irgb6 loading onto the E. cuniculi PVM at different time points post infection. 100 vacuoles were evaluated per sample, a black dot indicates that sample was not counted, three independent experiments are shown.

Mentions: When T. gondii infects IFNγ-treated mouse fibroblasts, the induced IRG proteins, especially the effector GKS proteins, accumulate on the PVM and lead to disruption of the vacuole [20]. To examine whether similar IRG-related processes might also occur on the microsporidian vacuole, we co-stained IFNγ-treated, E. cuniculi-infected, MEF cells with immunological reagents against individual GKS effector proteins (Irga6, Irgb6 and Irgd) as well against the GMS regulator proteins (Irgm1 and Irgm2) 24 h post infection (Figure 2). Some meronts were indeed coated with IRG proteins, but most were IRG-negative. Both Irga6-coated and uncoated vacuoles were found together in multiply infected host cells (Figure 2A). Irga6 and Irgb6 were found on vacuoles at higher frequencies, while Irgd and Irgm2 were found at lower but consistent frequencies (below 5%) (Figure 2B–E). Irgm1 was never found at the E. cuniculi PVM (more than 1000 meronts in three independent experiments were analysed). In IFNγ-induced cells, cytoplasmic Irga6 is predominantly in the GDP-bound form, but accumulates on the T. gondii PVM in the GTP-bound activated form, which can be specifically detected with the mouse antibody10D7 [17]. Because we could not conduct a co-staining with the mouse anti-meront antibody, 6G2, in combination with 10D7, we identified the meront via its enhanced DAPI signal to show that indeed Irga6 was accumulating on E. cuniculi vacuoles in the GTP-bound state (Figure 2F) as in T. gondii immunity. The number of vacuoles positive for Irga6 or Irgb6 was examined in more detail at different time points after infection (Figure 2G). The frequency of Irga6- and Irgb6-positive vacuoles varied between experiments (1–20%), but did not significantly increase or decrease between 0.5–24 hours post infection (Figure 2G) as the number of meronts progressively dropped, suggesting relatively fast clearance of the IRG-positive vacuoles.


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)

IRG proteins accumulate at the E. cuniculi parasitophorous vacuolar membrane.(A–F) MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 24 h. Fixed cells were stained for anti-meront mAB 6G2 as well as for endogenous Irga6 (165/3 pAS and 10D7 mAB), Irgb6 (A20 pAB), Irgd (2078 pAS) and Irgm2 (H53 pAS). Nuclei were labeled with DAPI. Representative microscopic images of IRG-positive E. cuniculi PVMs are presented. Yellow arrows point at the IRG-loaded PVM which is magnified at the end of each panel (zoom in the following order: upper left: merged image, upper right: phase contrast, lower left: anti-meront, lower right anti-IRG); white arrow: unloaded meront, every scale bar is 10 µm. (G) Quantification of Irga6 and Irgb6 loading onto the E. cuniculi PVM at different time points post infection. 100 vacuoles were evaluated per sample, a black dot indicates that sample was not counted, three independent experiments are shown.
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ppat-1004449-g002: IRG proteins accumulate at the E. cuniculi parasitophorous vacuolar membrane.(A–F) MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 24 h. Fixed cells were stained for anti-meront mAB 6G2 as well as for endogenous Irga6 (165/3 pAS and 10D7 mAB), Irgb6 (A20 pAB), Irgd (2078 pAS) and Irgm2 (H53 pAS). Nuclei were labeled with DAPI. Representative microscopic images of IRG-positive E. cuniculi PVMs are presented. Yellow arrows point at the IRG-loaded PVM which is magnified at the end of each panel (zoom in the following order: upper left: merged image, upper right: phase contrast, lower left: anti-meront, lower right anti-IRG); white arrow: unloaded meront, every scale bar is 10 µm. (G) Quantification of Irga6 and Irgb6 loading onto the E. cuniculi PVM at different time points post infection. 100 vacuoles were evaluated per sample, a black dot indicates that sample was not counted, three independent experiments are shown.
Mentions: When T. gondii infects IFNγ-treated mouse fibroblasts, the induced IRG proteins, especially the effector GKS proteins, accumulate on the PVM and lead to disruption of the vacuole [20]. To examine whether similar IRG-related processes might also occur on the microsporidian vacuole, we co-stained IFNγ-treated, E. cuniculi-infected, MEF cells with immunological reagents against individual GKS effector proteins (Irga6, Irgb6 and Irgd) as well against the GMS regulator proteins (Irgm1 and Irgm2) 24 h post infection (Figure 2). Some meronts were indeed coated with IRG proteins, but most were IRG-negative. Both Irga6-coated and uncoated vacuoles were found together in multiply infected host cells (Figure 2A). Irga6 and Irgb6 were found on vacuoles at higher frequencies, while Irgd and Irgm2 were found at lower but consistent frequencies (below 5%) (Figure 2B–E). Irgm1 was never found at the E. cuniculi PVM (more than 1000 meronts in three independent experiments were analysed). In IFNγ-induced cells, cytoplasmic Irga6 is predominantly in the GDP-bound form, but accumulates on the T. gondii PVM in the GTP-bound activated form, which can be specifically detected with the mouse antibody10D7 [17]. Because we could not conduct a co-staining with the mouse anti-meront antibody, 6G2, in combination with 10D7, we identified the meront via its enhanced DAPI signal to show that indeed Irga6 was accumulating on E. cuniculi vacuoles in the GTP-bound state (Figure 2F) as in T. gondii immunity. The number of vacuoles positive for Irga6 or Irgb6 was examined in more detail at different time points after infection (Figure 2G). The frequency of Irga6- and Irgb6-positive vacuoles varied between experiments (1–20%), but did not significantly increase or decrease between 0.5–24 hours post infection (Figure 2G) as the number of meronts progressively dropped, suggesting relatively fast clearance of the IRG-positive vacuoles.

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