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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 load onto PVM in a cooperative manner.MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 12 h (A) or 24 h (B, C). Fixed cells were stained for meronts (mouse mAB 6G2, red) as well as for endogenous Irga6 (rabbit pAS165/3, green) and Irgb6 (goat pAB A20, far-red pseudocolored in magenta). Nuclei were labeled with DAPI. Representative images from 4 independent experiments are shown; white box indicates enlarged area shown below; scale bar: 10 µm. (D) Quantification of cooperative loading after 24 h; Irgb6-single, Irga6-single or Irgb6/Irga6-double (both) positive meronts are shown as % of total 6G2-positive meronts; 100 vacuoles were counted in each independent experiment (Exp. 1–3).
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ppat-1004449-g003: IRG proteins load onto PVM in a cooperative manner.MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 12 h (A) or 24 h (B, C). Fixed cells were stained for meronts (mouse mAB 6G2, red) as well as for endogenous Irga6 (rabbit pAS165/3, green) and Irgb6 (goat pAB A20, far-red pseudocolored in magenta). Nuclei were labeled with DAPI. Representative images from 4 independent experiments are shown; white box indicates enlarged area shown below; scale bar: 10 µm. (D) Quantification of cooperative loading after 24 h; Irgb6-single, Irga6-single or Irgb6/Irga6-double (both) positive meronts are shown as % of total 6G2-positive meronts; 100 vacuoles were counted in each independent experiment (Exp. 1–3).

Mentions: A detailed view of IRG loading onto the T. gondii PVM has been established. IRG proteins accumulate on the PVM in a hierarchical order with Irgb6, Irgb10 and Irga6 as pioneers and demonstrate cooperative behaviour by stabilizing each other at the PVM [18]. In order to investigate cooperative loading on the E. cuniculi PVM, we conducted triple immunofluorescent stainings to identify the meront and two GKS proteins, Irga6 and Irgb6. Individual vacuoles positive for both IRG proteins were observed at early and late time points, such as 12 h post infection (Figure 3A) and 24 h post infection (Figure 3B, C). In most cases, Irga6 and Irgb6 co-localised to single vacuoles (Figure 3B), although often without accurate spatial coincidence on the PVM (Figure 3C). Notably, the number of E. cuniculi vacuoles accumulating both IRG proteins was higher than single-coated ones (Figure 3D). In view of the low frequencies of accumulation of individual IRG proteins, it is clear that the frequency of double-loaded vacuoles is highly non-random, suggesting that loading is cooperative between different IRG proteins, as seen on the T. gondii PVM.


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 load onto PVM in a cooperative manner.MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 12 h (A) or 24 h (B, C). Fixed cells were stained for meronts (mouse mAB 6G2, red) as well as for endogenous Irga6 (rabbit pAS165/3, green) and Irgb6 (goat pAB A20, far-red pseudocolored in magenta). Nuclei were labeled with DAPI. Representative images from 4 independent experiments are shown; white box indicates enlarged area shown below; scale bar: 10 µm. (D) Quantification of cooperative loading after 24 h; Irgb6-single, Irga6-single or Irgb6/Irga6-double (both) positive meronts are shown as % of total 6G2-positive meronts; 100 vacuoles were counted in each independent experiment (Exp. 1–3).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004449-g003: IRG proteins load onto PVM in a cooperative manner.MEFs were induced with IFNγ for 24 h and then infected with E. cuniculi spores for 12 h (A) or 24 h (B, C). Fixed cells were stained for meronts (mouse mAB 6G2, red) as well as for endogenous Irga6 (rabbit pAS165/3, green) and Irgb6 (goat pAB A20, far-red pseudocolored in magenta). Nuclei were labeled with DAPI. Representative images from 4 independent experiments are shown; white box indicates enlarged area shown below; scale bar: 10 µm. (D) Quantification of cooperative loading after 24 h; Irgb6-single, Irga6-single or Irgb6/Irga6-double (both) positive meronts are shown as % of total 6G2-positive meronts; 100 vacuoles were counted in each independent experiment (Exp. 1–3).
Mentions: A detailed view of IRG loading onto the T. gondii PVM has been established. IRG proteins accumulate on the PVM in a hierarchical order with Irgb6, Irgb10 and Irga6 as pioneers and demonstrate cooperative behaviour by stabilizing each other at the PVM [18]. In order to investigate cooperative loading on the E. cuniculi PVM, we conducted triple immunofluorescent stainings to identify the meront and two GKS proteins, Irga6 and Irgb6. Individual vacuoles positive for both IRG proteins were observed at early and late time points, such as 12 h post infection (Figure 3A) and 24 h post infection (Figure 3B, C). In most cases, Irga6 and Irgb6 co-localised to single vacuoles (Figure 3B), although often without accurate spatial coincidence on the PVM (Figure 3C). Notably, the number of E. cuniculi vacuoles accumulating both IRG proteins was higher than single-coated ones (Figure 3D). In view of the low frequencies of accumulation of individual IRG proteins, it is clear that the frequency of double-loaded vacuoles is highly non-random, suggesting that loading is cooperative between different IRG proteins, as seen on the T. gondii PVM.

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