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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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IRG proteins load in a consistent hierarchy on to the PV of T. gondii ME49 strain. A. Each IRG protein loads onto a characteristic proportion of vacuoles. Quantification of IRG-positive PVs (%) observed in IFNγ-induced 2 h T. gondii ME49 infected MEFs and gs3T3 cells assayed by immunocytochemistry using antibody reagents described in Fig. S4 and in Experimental procedures. At least three independent experiments were assayed and pooled and a minimum of 500 PVs counted for each IRG protein (error bars indicate the standard deviation between individual experiments). The statistical significances of the differences recorded were determined by Student's t-test and are shown on the figure (***P < 0.001; *P < 0.05). B. IRG proteins do not load at random onto each vacuole. IRG proteins loaded onto T. gondii PV were detected by co-staining with pairs of specific antibodies directed against IRG proteins at different positions in the hierarchy, using specific secondary reagents carrying different fluorochromes. Vacuoles loaded with one IRG protein were scored for possession of the second and vice versa. Vacuoles loaded with neither IRG protein were not included in the analysis. At least 100 positive vacuoles were counted for each pair of IRG proteins. Red bar segments give the percentage of vacuoles loaded with both IRG proteins in a given pair, while the green and black bar segments give respectively the percentages loaded with only the lower or only the higher member. The PV loading of pairs of IRG proteins is very strongly correlated such that nearly every vacuole loaded with an IRG protein lower down the hierarchy is also loaded with an IRG protein higher in the hierarchy. The full data are shown in Table S1. C. Irgb6 loads more heavily onto T. gondii vacuoles at early time points than Irga6. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii ME49 strain. At indicated times after infection Irgb6 and Irga6 vacuole loading intensities were analysed simultaneously with specific primary antibodies (Irgb6, serum A20; Irga6, mAb 10D7) detected with secondary antibodies labelled with different fluorochromes. D. Irgb6 loads before Irgd on to the T. gondii ME49 strain PV. C57BL/6 MEFs were induced with IFNγ and transfected simultaneously with constructs expressing Irgb6-FLAG-EGFP and Irgd-ctag1-Cherry. After 24 h, cells were infected with T. gondii ME49 strain in microscope slide chambers and observed by live cell imaging for the accumulation of IRG proteins. Successive 1 min frames from one vacuole show Irgb6-FLAG-EGFP visibly loading several minutes before Irgd-ctag1-Cherry. E. Absence of Irga6 does not affect the proportion of vacuoles loaded with Irgb6 or Irgd. Irga6−/− and wt MEFs were induced with IFNγ and infected with T. gondii strain ME49. 2 h after infection cells were stained with appropriate antibody reagents and the proportion of Irgb6 (mAb B34) and Irgd (serum 081/1) labelled vacuoles (out of 300 for each IRG protein in two independent experiments) was recorded. F. Intensity of PV loading by Irgb6 is significantly reduced in Irga6−/− relative to wt MEFs (P < 0.001 by Mann–Whitney test, indicated by *** on the figure). IFNγ-induced Irga6−/− and wt MEFs were infected with T. gondii ME49 strain. Two hours after infection slides were stained for Irgb6 (B34), Irgd (081/1) and Irgm2 (H53/3). At least 50 vacuoles loaded with each IRG protein were assayed for loading intensity from both cell types. The arithmetic means are given as horizontal lines.
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fig06: IRG proteins load in a consistent hierarchy on to the PV of T. gondii ME49 strain. A. Each IRG protein loads onto a characteristic proportion of vacuoles. Quantification of IRG-positive PVs (%) observed in IFNγ-induced 2 h T. gondii ME49 infected MEFs and gs3T3 cells assayed by immunocytochemistry using antibody reagents described in Fig. S4 and in Experimental procedures. At least three independent experiments were assayed and pooled and a minimum of 500 PVs counted for each IRG protein (error bars indicate the standard deviation between individual experiments). The statistical significances of the differences recorded were determined by Student's t-test and are shown on the figure (***P < 0.001; *P < 0.05). B. IRG proteins do not load at random onto each vacuole. IRG proteins loaded onto T. gondii PV were detected by co-staining with pairs of specific antibodies directed against IRG proteins at different positions in the hierarchy, using specific secondary reagents carrying different fluorochromes. Vacuoles loaded with one IRG protein were scored for possession of the second and vice versa. Vacuoles loaded with neither IRG protein were not included in the analysis. At least 100 positive vacuoles were counted for each pair of IRG proteins. Red bar segments give the percentage of vacuoles loaded with both IRG proteins in a given pair, while the green and black bar segments give respectively the percentages loaded with only the lower or only the higher member. The PV loading of pairs of IRG proteins is very strongly correlated such that nearly every vacuole loaded with an IRG protein lower down the hierarchy is also loaded with an IRG protein higher in the hierarchy. The full data are shown in Table S1. C. Irgb6 loads more heavily onto T. gondii vacuoles at early time points than Irga6. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii ME49 strain. At indicated times after infection Irgb6 and Irga6 vacuole loading intensities were analysed simultaneously with specific primary antibodies (Irgb6, serum A20; Irga6, mAb 10D7) detected with secondary antibodies labelled with different fluorochromes. D. Irgb6 loads before Irgd on to the T. gondii ME49 strain PV. C57BL/6 MEFs were induced with IFNγ and transfected simultaneously with constructs expressing Irgb6-FLAG-EGFP and Irgd-ctag1-Cherry. After 24 h, cells were infected with T. gondii ME49 strain in microscope slide chambers and observed by live cell imaging for the accumulation of IRG proteins. Successive 1 min frames from one vacuole show Irgb6-FLAG-EGFP visibly loading several minutes before Irgd-ctag1-Cherry. E. Absence of Irga6 does not affect the proportion of vacuoles loaded with Irgb6 or Irgd. Irga6−/− and wt MEFs were induced with IFNγ and infected with T. gondii strain ME49. 2 h after infection cells were stained with appropriate antibody reagents and the proportion of Irgb6 (mAb B34) and Irgd (serum 081/1) labelled vacuoles (out of 300 for each IRG protein in two independent experiments) was recorded. F. Intensity of PV loading by Irgb6 is significantly reduced in Irga6−/− relative to wt MEFs (P < 0.001 by Mann–Whitney test, indicated by *** on the figure). IFNγ-induced Irga6−/− and wt MEFs were infected with T. gondii ME49 strain. Two hours after infection slides were stained for Irgb6 (B34), Irgd (081/1) and Irgm2 (H53/3). At least 50 vacuoles loaded with each IRG protein were assayed for loading intensity from both cell types. The arithmetic means are given as horizontal lines.

Mentions: Each IRG protein accumulates on a characteristic proportion of vacuoles. Thus Irga6 and Irgb6 both load onto the majority of vacuoles (Figs 1–5) (Martens et al., 2005; Hunn et al., 2008; Papic et al., 2008), but Irgb6 invariably loads onto a higher proportion of vacuoles than Irga6 (Figs 4 and 5A). By discriminating two or three IRG proteins at a time on single intracellular parasites we examined the proportions of loaded vacuoles, as well as the coupled or uncoupled distribution, of Irga6, Irgb6, Irgb10, Irgd, Irgm2 and Irgm3. All antibody reagents used showed saturation binding to their vacuolar targets (Fig. S4 and A. Khaminets and S. Könen-Waisman, unpublished data). Four different immunoreagents failed to find any vacuolar loading of Irgm1, despite its strong association with resistance to T. gondii (S. Könen-Waisman, unpublished data and see also Martens et al., 2005). Vacuolar loading by five IRG proteins fell into a consistent hierarchy in the order Irgb6 > Irgb10 > Irga6 > Irgm2 ≈ Irgd (Fig. 6A). Irgm3 frequency was low, probably below Irgd/Irgm2, but a value is not shown because the loading intensity was low and difficult to resolve from the surrounding ER. The hierarchy was reproducible except for the relative positions of Irgd and Irgm2 and was independent of the reagents used.


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)

IRG proteins load in a consistent hierarchy on to the PV of T. gondii ME49 strain. A. Each IRG protein loads onto a characteristic proportion of vacuoles. Quantification of IRG-positive PVs (%) observed in IFNγ-induced 2 h T. gondii ME49 infected MEFs and gs3T3 cells assayed by immunocytochemistry using antibody reagents described in Fig. S4 and in Experimental procedures. At least three independent experiments were assayed and pooled and a minimum of 500 PVs counted for each IRG protein (error bars indicate the standard deviation between individual experiments). The statistical significances of the differences recorded were determined by Student's t-test and are shown on the figure (***P < 0.001; *P < 0.05). B. IRG proteins do not load at random onto each vacuole. IRG proteins loaded onto T. gondii PV were detected by co-staining with pairs of specific antibodies directed against IRG proteins at different positions in the hierarchy, using specific secondary reagents carrying different fluorochromes. Vacuoles loaded with one IRG protein were scored for possession of the second and vice versa. Vacuoles loaded with neither IRG protein were not included in the analysis. At least 100 positive vacuoles were counted for each pair of IRG proteins. Red bar segments give the percentage of vacuoles loaded with both IRG proteins in a given pair, while the green and black bar segments give respectively the percentages loaded with only the lower or only the higher member. The PV loading of pairs of IRG proteins is very strongly correlated such that nearly every vacuole loaded with an IRG protein lower down the hierarchy is also loaded with an IRG protein higher in the hierarchy. The full data are shown in Table S1. C. Irgb6 loads more heavily onto T. gondii vacuoles at early time points than Irga6. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii ME49 strain. At indicated times after infection Irgb6 and Irga6 vacuole loading intensities were analysed simultaneously with specific primary antibodies (Irgb6, serum A20; Irga6, mAb 10D7) detected with secondary antibodies labelled with different fluorochromes. D. Irgb6 loads before Irgd on to the T. gondii ME49 strain PV. C57BL/6 MEFs were induced with IFNγ and transfected simultaneously with constructs expressing Irgb6-FLAG-EGFP and Irgd-ctag1-Cherry. After 24 h, cells were infected with T. gondii ME49 strain in microscope slide chambers and observed by live cell imaging for the accumulation of IRG proteins. Successive 1 min frames from one vacuole show Irgb6-FLAG-EGFP visibly loading several minutes before Irgd-ctag1-Cherry. E. Absence of Irga6 does not affect the proportion of vacuoles loaded with Irgb6 or Irgd. Irga6−/− and wt MEFs were induced with IFNγ and infected with T. gondii strain ME49. 2 h after infection cells were stained with appropriate antibody reagents and the proportion of Irgb6 (mAb B34) and Irgd (serum 081/1) labelled vacuoles (out of 300 for each IRG protein in two independent experiments) was recorded. F. Intensity of PV loading by Irgb6 is significantly reduced in Irga6−/− relative to wt MEFs (P < 0.001 by Mann–Whitney test, indicated by *** on the figure). IFNγ-induced Irga6−/− and wt MEFs were infected with T. gondii ME49 strain. Two hours after infection slides were stained for Irgb6 (B34), Irgd (081/1) and Irgm2 (H53/3). At least 50 vacuoles loaded with each IRG protein were assayed for loading intensity from both cell types. The arithmetic means are given as horizontal lines.
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fig06: IRG proteins load in a consistent hierarchy on to the PV of T. gondii ME49 strain. A. Each IRG protein loads onto a characteristic proportion of vacuoles. Quantification of IRG-positive PVs (%) observed in IFNγ-induced 2 h T. gondii ME49 infected MEFs and gs3T3 cells assayed by immunocytochemistry using antibody reagents described in Fig. S4 and in Experimental procedures. At least three independent experiments were assayed and pooled and a minimum of 500 PVs counted for each IRG protein (error bars indicate the standard deviation between individual experiments). The statistical significances of the differences recorded were determined by Student's t-test and are shown on the figure (***P < 0.001; *P < 0.05). B. IRG proteins do not load at random onto each vacuole. IRG proteins loaded onto T. gondii PV were detected by co-staining with pairs of specific antibodies directed against IRG proteins at different positions in the hierarchy, using specific secondary reagents carrying different fluorochromes. Vacuoles loaded with one IRG protein were scored for possession of the second and vice versa. Vacuoles loaded with neither IRG protein were not included in the analysis. At least 100 positive vacuoles were counted for each pair of IRG proteins. Red bar segments give the percentage of vacuoles loaded with both IRG proteins in a given pair, while the green and black bar segments give respectively the percentages loaded with only the lower or only the higher member. The PV loading of pairs of IRG proteins is very strongly correlated such that nearly every vacuole loaded with an IRG protein lower down the hierarchy is also loaded with an IRG protein higher in the hierarchy. The full data are shown in Table S1. C. Irgb6 loads more heavily onto T. gondii vacuoles at early time points than Irga6. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii ME49 strain. At indicated times after infection Irgb6 and Irga6 vacuole loading intensities were analysed simultaneously with specific primary antibodies (Irgb6, serum A20; Irga6, mAb 10D7) detected with secondary antibodies labelled with different fluorochromes. D. Irgb6 loads before Irgd on to the T. gondii ME49 strain PV. C57BL/6 MEFs were induced with IFNγ and transfected simultaneously with constructs expressing Irgb6-FLAG-EGFP and Irgd-ctag1-Cherry. After 24 h, cells were infected with T. gondii ME49 strain in microscope slide chambers and observed by live cell imaging for the accumulation of IRG proteins. Successive 1 min frames from one vacuole show Irgb6-FLAG-EGFP visibly loading several minutes before Irgd-ctag1-Cherry. E. Absence of Irga6 does not affect the proportion of vacuoles loaded with Irgb6 or Irgd. Irga6−/− and wt MEFs were induced with IFNγ and infected with T. gondii strain ME49. 2 h after infection cells were stained with appropriate antibody reagents and the proportion of Irgb6 (mAb B34) and Irgd (serum 081/1) labelled vacuoles (out of 300 for each IRG protein in two independent experiments) was recorded. F. Intensity of PV loading by Irgb6 is significantly reduced in Irga6−/− relative to wt MEFs (P < 0.001 by Mann–Whitney test, indicated by *** on the figure). IFNγ-induced Irga6−/− and wt MEFs were infected with T. gondii ME49 strain. Two hours after infection slides were stained for Irgb6 (B34), Irgd (081/1) and Irgm2 (H53/3). At least 50 vacuoles loaded with each IRG protein were assayed for loading intensity from both cell types. The arithmetic means are given as horizontal lines.
Mentions: Each IRG protein accumulates on a characteristic proportion of vacuoles. Thus Irga6 and Irgb6 both load onto the majority of vacuoles (Figs 1–5) (Martens et al., 2005; Hunn et al., 2008; Papic et al., 2008), but Irgb6 invariably loads onto a higher proportion of vacuoles than Irga6 (Figs 4 and 5A). By discriminating two or three IRG proteins at a time on single intracellular parasites we examined the proportions of loaded vacuoles, as well as the coupled or uncoupled distribution, of Irga6, Irgb6, Irgb10, Irgd, Irgm2 and Irgm3. All antibody reagents used showed saturation binding to their vacuolar targets (Fig. S4 and A. Khaminets and S. Könen-Waisman, unpublished data). Four different immunoreagents failed to find any vacuolar loading of Irgm1, despite its strong association with resistance to T. gondii (S. Könen-Waisman, unpublished data and see also Martens et al., 2005). Vacuolar loading by five IRG proteins fell into a consistent hierarchy in the order Irgb6 > Irgb10 > Irga6 > Irgm2 ≈ Irgd (Fig. 6A). Irgm3 frequency was low, probably below Irgd/Irgm2, but a value is not shown because the loading intensity was low and difficult to resolve from the surrounding ER. The hierarchy was reproducible except for the relative positions of Irgd and Irgm2 and was independent of the reagents used.

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