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Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole.

Khaminets A, Hunn JP, Könen-Waisman S, Zhao YO, Preukschat D, Coers J, Boyle JP, Ong YC, Boothroyd JC, Reichmann G, Howard JC - Cell. Microbiol. (2010)

Bottom Line: Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected.The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect.The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Zülpicher Strasse, Cologne 50674, Germany.

ABSTRACT
The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

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Influence of duration of IFNγ induction on Irga6 and Irgb6 protein levels and on vacuolar loading. MEFs were induced for different times with IFNγ before infection with T. gondii strain ME49 for 2 h and stained in immunofluorescence against Irga6 and Irgb6. Co-staining against GRA7 was used to determine intracellular parasites. A. and B. The pixel intensities of (A) Irgb6 (serum A20) and (B) Irga6 (serum 165) signals at the PVM of ME49 vacuoles were determined as described in Experimental procedures (see also Fig. S1) and displayed as a function of IFNγ induction time. Sixty PVs were quantified per time point and the arithmetic means are given as horizontal lines. C. In parallel sample cell lysates from MEFs induced for the indicated times with IFNγ were analysed by Western blot for Irga6 (mAb 10D7) and Irgb6 (mAb B34) expression level relative to calnexin as a loading control.
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fig03: Influence of duration of IFNγ induction on Irga6 and Irgb6 protein levels and on vacuolar loading. MEFs were induced for different times with IFNγ before infection with T. gondii strain ME49 for 2 h and stained in immunofluorescence against Irga6 and Irgb6. Co-staining against GRA7 was used to determine intracellular parasites. A. and B. The pixel intensities of (A) Irgb6 (serum A20) and (B) Irga6 (serum 165) signals at the PVM of ME49 vacuoles were determined as described in Experimental procedures (see also Fig. S1) and displayed as a function of IFNγ induction time. Sixty PVs were quantified per time point and the arithmetic means are given as horizontal lines. C. In parallel sample cell lysates from MEFs induced for the indicated times with IFNγ were analysed by Western blot for Irga6 (mAb 10D7) and Irgb6 (mAb B34) expression level relative to calnexin as a loading control.

Mentions: The combined intensities of vacuolar accumulation recorded for Irga6 and Irgb6 during the first 2 h after infection were remarkably heterogeneous (Fig. 1C). We analysed pixel intensities of Irga6 and Irgb6 independently at the PVM in MEFs induced for 12–48 h with IFN and infected for a further 2 h with T. gondii (Fig. 3A and B). Absolute levels of Irgb6 and to a lesser extent Irga6 protein in the cells increased with time after IFNγ stimulation (Fig. 3C). Sixty intracellular vacuoles were quantified per time point for Irga6 and Irgb6. The great majority of vacuoles accumulated some IRG protein but the amount accumulated varied from very high values all the way down to the visible threshold and below. The mean expression of Irga6 and Irgb6 increased up to 48 h after IFN induction (Fig. 3C) as did the IRG protein level on the 2 h post-infection PVM (Fig. 3A and B), suggesting that IRG protein accumulation on the vacuole is partly concentration-driven. So heterogeneity in IRG protein level in individual cells could contribute to heterogeneity of PVM loading. Nevertheless most of the observed heterogeneity is evidently intrinsic to the individual vacuole. This could be visualized directly for the cell shown in Fig. 2B (Video S2), which is already infected with one T. gondii (arrow) before a second one enters (arrowhead). The PVM of the first parasite already has clear Irga6-ctag1-EGFP accumulation that does not change during the video while the accumulation on the PVM of the second T. gondii rapidly overhauls the first one and becomes very bright. Some vacuoles remained free of IRG protein for hours: other vacuoles in the same cell could be heavily loaded. The nature of this vacuole-specific heterogeneity in IRG loading is obviously of interest as it relates directly to the ability of individual parasites to escape attack by IRG resistance proteins.


Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole.

Khaminets A, Hunn JP, Könen-Waisman S, Zhao YO, Preukschat D, Coers J, Boyle JP, Ong YC, Boothroyd JC, Reichmann G, Howard JC - Cell. Microbiol. (2010)

Influence of duration of IFNγ induction on Irga6 and Irgb6 protein levels and on vacuolar loading. MEFs were induced for different times with IFNγ before infection with T. gondii strain ME49 for 2 h and stained in immunofluorescence against Irga6 and Irgb6. Co-staining against GRA7 was used to determine intracellular parasites. A. and B. The pixel intensities of (A) Irgb6 (serum A20) and (B) Irga6 (serum 165) signals at the PVM of ME49 vacuoles were determined as described in Experimental procedures (see also Fig. S1) and displayed as a function of IFNγ induction time. Sixty PVs were quantified per time point and the arithmetic means are given as horizontal lines. C. In parallel sample cell lysates from MEFs induced for the indicated times with IFNγ were analysed by Western blot for Irga6 (mAb 10D7) and Irgb6 (mAb B34) expression level relative to calnexin as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2901525&req=5

fig03: Influence of duration of IFNγ induction on Irga6 and Irgb6 protein levels and on vacuolar loading. MEFs were induced for different times with IFNγ before infection with T. gondii strain ME49 for 2 h and stained in immunofluorescence against Irga6 and Irgb6. Co-staining against GRA7 was used to determine intracellular parasites. A. and B. The pixel intensities of (A) Irgb6 (serum A20) and (B) Irga6 (serum 165) signals at the PVM of ME49 vacuoles were determined as described in Experimental procedures (see also Fig. S1) and displayed as a function of IFNγ induction time. Sixty PVs were quantified per time point and the arithmetic means are given as horizontal lines. C. In parallel sample cell lysates from MEFs induced for the indicated times with IFNγ were analysed by Western blot for Irga6 (mAb 10D7) and Irgb6 (mAb B34) expression level relative to calnexin as a loading control.
Mentions: The combined intensities of vacuolar accumulation recorded for Irga6 and Irgb6 during the first 2 h after infection were remarkably heterogeneous (Fig. 1C). We analysed pixel intensities of Irga6 and Irgb6 independently at the PVM in MEFs induced for 12–48 h with IFN and infected for a further 2 h with T. gondii (Fig. 3A and B). Absolute levels of Irgb6 and to a lesser extent Irga6 protein in the cells increased with time after IFNγ stimulation (Fig. 3C). Sixty intracellular vacuoles were quantified per time point for Irga6 and Irgb6. The great majority of vacuoles accumulated some IRG protein but the amount accumulated varied from very high values all the way down to the visible threshold and below. The mean expression of Irga6 and Irgb6 increased up to 48 h after IFN induction (Fig. 3C) as did the IRG protein level on the 2 h post-infection PVM (Fig. 3A and B), suggesting that IRG protein accumulation on the vacuole is partly concentration-driven. So heterogeneity in IRG protein level in individual cells could contribute to heterogeneity of PVM loading. Nevertheless most of the observed heterogeneity is evidently intrinsic to the individual vacuole. This could be visualized directly for the cell shown in Fig. 2B (Video S2), which is already infected with one T. gondii (arrow) before a second one enters (arrowhead). The PVM of the first parasite already has clear Irga6-ctag1-EGFP accumulation that does not change during the video while the accumulation on the PVM of the second T. gondii rapidly overhauls the first one and becomes very bright. Some vacuoles remained free of IRG protein for hours: other vacuoles in the same cell could be heavily loaded. The nature of this vacuole-specific heterogeneity in IRG loading is obviously of interest as it relates directly to the ability of individual parasites to escape attack by IRG resistance proteins.

Bottom Line: Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected.The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect.The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Institute for Genetics, University of Cologne, Zülpicher Strasse, Cologne 50674, Germany.

ABSTRACT
The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

Show MeSH
Related in: MedlinePlus