Limits...
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.

Show MeSH

Related in: MedlinePlus

Accumulation of IRG proteins on the PVM is reduced in virulent T. gondii infection. A. IFNγ-induced MEFs were infected for 2 h with type I virulent (RH and BK), type II (ME49 and NTE) and type III (CTG) avirulent T. gondii strains and assayed microscopically for Irgb6-positive vacuoles (serum A20). Irgb6-positive PVs were counted for each parasite strain from 400–600 intracellular parasites in two independent experiments and pooled. The differences between the avirulent types II and III strains and the virulent type I strains are highly significant by chi-squared test (***P < 0.001). B. gs3T3 cells were induced with IFNγ and infected with T. gondii ME49 strain (black bars) or RH-YFP strain (white bars). Numbers of Irgb6-, Irgb10-, Irga6- and Irgd-positive PVs were counted in 3–6 experiments for each IRG protein and T. gondii strain and given as a percentage of intracellular parasites. More than 200 intracellular parasites were counted blind per experiment. The mean percentages of positive vacuoles of ME49 or RH-YFP type for each IRG protein are shown. Error bars indicate the standard deviations. The significances of the differences between loading of ME49 and RH-YFP vacuoles are given on the figure (***P < 0.001, **P < 0.01, *P < 0.05, by Student's t-test). C. C57BL/6 MEFs were induced with IFNγ and infected with RH-YFP. Irgb6 (blue) and Irga6 (red) were detected in immunofluorescence with serum A20 and mAb 10E7 respectively. Intracellular fluorescent parasites (RH-YFP, green) identified in phase contrast (PhC) are indicated by white arrowheads (strongly IRG-positive) and arrows (weakly IRG-positive). D. gs3T3 fibroblasts were induced with IFNγ and infected with either ME49 or RH-YFP T. gondii strains. Mean fluorescence intensities of Irga6 (serum 165/3) and Irgb6 (serum A20) signals at the PVM were quantified as described in Fig. S1 and Experimental procedures. Thirty-five random PVs per data set were quantified blind. For both Irga6 and Irgb6, the different loading intensities on ME49 and RH-YFP vacuoles were highly significant (***P < 0.001). E. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii RH-YFP strain. Mean fluorescence intensities of Irga6 and Irgb6 were measured for selected PVs expressing no detectable (open circles), weak (grey filled circles) or strong Irga6 staining (black filled circles). The fluorescent intensity profiles of five representative PVs per group are displayed in Fig. S6. F. Photomicrograph of an IFNγ-stimulated MEF shown 2 h after double infection with ME49 strain (indicated by arrowhead) and RH-YFP strain T. gondii (green, indicated by arrow). The ME49 strain parasite shows intense Irgb6 (serum A20, red) accumulation at the PV while the RH-YFP in the same cell has no Irgb6 on the PV. G. IFNγ-stimulated MEFs were infected with T. gondii ME49 strain alone or simultaneously with ME49 and RH-YFP strains. Irgb6 (detected by serum A20) and Irga6 (detected by mAb 10D7) fluorescence intensities were measured on at least 30 ME49 PVs in singly and doubly infected cells. ME49 and RH-YFP were discriminated by the fluorescent signal from RH-YFP. The arithmetic means are given as horizontal lines. The loading of Irga6 and Irgb6 onto PVs of avirulent ME49 strain T. gondii was unaffected by the simultaneous presence of virulent RH-YFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2901525&req=5

fig08: Accumulation of IRG proteins on the PVM is reduced in virulent T. gondii infection. A. IFNγ-induced MEFs were infected for 2 h with type I virulent (RH and BK), type II (ME49 and NTE) and type III (CTG) avirulent T. gondii strains and assayed microscopically for Irgb6-positive vacuoles (serum A20). Irgb6-positive PVs were counted for each parasite strain from 400–600 intracellular parasites in two independent experiments and pooled. The differences between the avirulent types II and III strains and the virulent type I strains are highly significant by chi-squared test (***P < 0.001). B. gs3T3 cells were induced with IFNγ and infected with T. gondii ME49 strain (black bars) or RH-YFP strain (white bars). Numbers of Irgb6-, Irgb10-, Irga6- and Irgd-positive PVs were counted in 3–6 experiments for each IRG protein and T. gondii strain and given as a percentage of intracellular parasites. More than 200 intracellular parasites were counted blind per experiment. The mean percentages of positive vacuoles of ME49 or RH-YFP type for each IRG protein are shown. Error bars indicate the standard deviations. The significances of the differences between loading of ME49 and RH-YFP vacuoles are given on the figure (***P < 0.001, **P < 0.01, *P < 0.05, by Student's t-test). C. C57BL/6 MEFs were induced with IFNγ and infected with RH-YFP. Irgb6 (blue) and Irga6 (red) were detected in immunofluorescence with serum A20 and mAb 10E7 respectively. Intracellular fluorescent parasites (RH-YFP, green) identified in phase contrast (PhC) are indicated by white arrowheads (strongly IRG-positive) and arrows (weakly IRG-positive). D. gs3T3 fibroblasts were induced with IFNγ and infected with either ME49 or RH-YFP T. gondii strains. Mean fluorescence intensities of Irga6 (serum 165/3) and Irgb6 (serum A20) signals at the PVM were quantified as described in Fig. S1 and Experimental procedures. Thirty-five random PVs per data set were quantified blind. For both Irga6 and Irgb6, the different loading intensities on ME49 and RH-YFP vacuoles were highly significant (***P < 0.001). E. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii RH-YFP strain. Mean fluorescence intensities of Irga6 and Irgb6 were measured for selected PVs expressing no detectable (open circles), weak (grey filled circles) or strong Irga6 staining (black filled circles). The fluorescent intensity profiles of five representative PVs per group are displayed in Fig. S6. F. Photomicrograph of an IFNγ-stimulated MEF shown 2 h after double infection with ME49 strain (indicated by arrowhead) and RH-YFP strain T. gondii (green, indicated by arrow). The ME49 strain parasite shows intense Irgb6 (serum A20, red) accumulation at the PV while the RH-YFP in the same cell has no Irgb6 on the PV. G. IFNγ-stimulated MEFs were infected with T. gondii ME49 strain alone or simultaneously with ME49 and RH-YFP strains. Irgb6 (detected by serum A20) and Irga6 (detected by mAb 10D7) fluorescence intensities were measured on at least 30 ME49 PVs in singly and doubly infected cells. ME49 and RH-YFP were discriminated by the fluorescent signal from RH-YFP. The arithmetic means are given as horizontal lines. The loading of Irga6 and Irgb6 onto PVs of avirulent ME49 strain T. gondii was unaffected by the simultaneous presence of virulent RH-YFP.

Mentions: Reduced loading of Irgb6 on virulent type I strain RH PVs has recently been reported in MEFs and macrophages, correlating with reduced vacuolar disruption (Zhao et al., 2009a,b,c;). The data shown above were all based on infection of cells with the avirulent T. gondii strain, ME49. As shown in Fig. 8A, two type I strains, BK and RH, gave grossly defective loading of Irgb6, while two avirulent type II strains, ME49 and NTE, and an avirulent type III strain, CTG, showed the familiar high frequency of Irgb6-loaded vacuoles. Loading of all IRG proteins was attenuated on RH strain vacuoles (Fig. 8B) but two different effects were apparent. Irgb6 and Irgb10 loaded onto very few vacuoles, while for Irga6 or Irgd the number of loaded vacuoles was significantly but not dramatically reduced but the amount loaded per vacuole was generally much lower, documented for Irga6 in Figs 8C and D and S6. The few vacuoles loaded with Irgb6 were loaded very heavily (Fig. 8C and D) and were additionally all intensely loaded for Irga6 (Figs 8E and S6) and Irgd (J.P. Hunn and S. Könen-Waisman, unpublished data). In addition to their implications for the nature of virulence, these results also support the concept that Irgb6 and Irgb10 function as loading pioneers. In their absence, the remaining IRG proteins have difficulty gaining a foothold on the vacuole. In IFN-induced MEFs co-infected with RH-YFP and unlabelled ME49, ME49-containing vacuoles could be intensely coated with Irgb6 while RH-containing vacuoles in the same cell had none (Fig. 8F), and the loading intensity of Irgb6 and Irga6 on ME49 PVs was unaffected by the presence of RH-YFP vacuoles (Fig. 8G). It is therefore unlikely that a diffusible molecule from the virulent strain is responsible for reduced Irgb6 loading. Our results and conclusions confirm those of Zhao et al. (Zhao et al., 2009c).


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)

Accumulation of IRG proteins on the PVM is reduced in virulent T. gondii infection. A. IFNγ-induced MEFs were infected for 2 h with type I virulent (RH and BK), type II (ME49 and NTE) and type III (CTG) avirulent T. gondii strains and assayed microscopically for Irgb6-positive vacuoles (serum A20). Irgb6-positive PVs were counted for each parasite strain from 400–600 intracellular parasites in two independent experiments and pooled. The differences between the avirulent types II and III strains and the virulent type I strains are highly significant by chi-squared test (***P < 0.001). B. gs3T3 cells were induced with IFNγ and infected with T. gondii ME49 strain (black bars) or RH-YFP strain (white bars). Numbers of Irgb6-, Irgb10-, Irga6- and Irgd-positive PVs were counted in 3–6 experiments for each IRG protein and T. gondii strain and given as a percentage of intracellular parasites. More than 200 intracellular parasites were counted blind per experiment. The mean percentages of positive vacuoles of ME49 or RH-YFP type for each IRG protein are shown. Error bars indicate the standard deviations. The significances of the differences between loading of ME49 and RH-YFP vacuoles are given on the figure (***P < 0.001, **P < 0.01, *P < 0.05, by Student's t-test). C. C57BL/6 MEFs were induced with IFNγ and infected with RH-YFP. Irgb6 (blue) and Irga6 (red) were detected in immunofluorescence with serum A20 and mAb 10E7 respectively. Intracellular fluorescent parasites (RH-YFP, green) identified in phase contrast (PhC) are indicated by white arrowheads (strongly IRG-positive) and arrows (weakly IRG-positive). D. gs3T3 fibroblasts were induced with IFNγ and infected with either ME49 or RH-YFP T. gondii strains. Mean fluorescence intensities of Irga6 (serum 165/3) and Irgb6 (serum A20) signals at the PVM were quantified as described in Fig. S1 and Experimental procedures. Thirty-five random PVs per data set were quantified blind. For both Irga6 and Irgb6, the different loading intensities on ME49 and RH-YFP vacuoles were highly significant (***P < 0.001). E. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii RH-YFP strain. Mean fluorescence intensities of Irga6 and Irgb6 were measured for selected PVs expressing no detectable (open circles), weak (grey filled circles) or strong Irga6 staining (black filled circles). The fluorescent intensity profiles of five representative PVs per group are displayed in Fig. S6. F. Photomicrograph of an IFNγ-stimulated MEF shown 2 h after double infection with ME49 strain (indicated by arrowhead) and RH-YFP strain T. gondii (green, indicated by arrow). The ME49 strain parasite shows intense Irgb6 (serum A20, red) accumulation at the PV while the RH-YFP in the same cell has no Irgb6 on the PV. G. IFNγ-stimulated MEFs were infected with T. gondii ME49 strain alone or simultaneously with ME49 and RH-YFP strains. Irgb6 (detected by serum A20) and Irga6 (detected by mAb 10D7) fluorescence intensities were measured on at least 30 ME49 PVs in singly and doubly infected cells. ME49 and RH-YFP were discriminated by the fluorescent signal from RH-YFP. The arithmetic means are given as horizontal lines. The loading of Irga6 and Irgb6 onto PVs of avirulent ME49 strain T. gondii was unaffected by the simultaneous presence of virulent RH-YFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig08: Accumulation of IRG proteins on the PVM is reduced in virulent T. gondii infection. A. IFNγ-induced MEFs were infected for 2 h with type I virulent (RH and BK), type II (ME49 and NTE) and type III (CTG) avirulent T. gondii strains and assayed microscopically for Irgb6-positive vacuoles (serum A20). Irgb6-positive PVs were counted for each parasite strain from 400–600 intracellular parasites in two independent experiments and pooled. The differences between the avirulent types II and III strains and the virulent type I strains are highly significant by chi-squared test (***P < 0.001). B. gs3T3 cells were induced with IFNγ and infected with T. gondii ME49 strain (black bars) or RH-YFP strain (white bars). Numbers of Irgb6-, Irgb10-, Irga6- and Irgd-positive PVs were counted in 3–6 experiments for each IRG protein and T. gondii strain and given as a percentage of intracellular parasites. More than 200 intracellular parasites were counted blind per experiment. The mean percentages of positive vacuoles of ME49 or RH-YFP type for each IRG protein are shown. Error bars indicate the standard deviations. The significances of the differences between loading of ME49 and RH-YFP vacuoles are given on the figure (***P < 0.001, **P < 0.01, *P < 0.05, by Student's t-test). C. C57BL/6 MEFs were induced with IFNγ and infected with RH-YFP. Irgb6 (blue) and Irga6 (red) were detected in immunofluorescence with serum A20 and mAb 10E7 respectively. Intracellular fluorescent parasites (RH-YFP, green) identified in phase contrast (PhC) are indicated by white arrowheads (strongly IRG-positive) and arrows (weakly IRG-positive). D. gs3T3 fibroblasts were induced with IFNγ and infected with either ME49 or RH-YFP T. gondii strains. Mean fluorescence intensities of Irga6 (serum 165/3) and Irgb6 (serum A20) signals at the PVM were quantified as described in Fig. S1 and Experimental procedures. Thirty-five random PVs per data set were quantified blind. For both Irga6 and Irgb6, the different loading intensities on ME49 and RH-YFP vacuoles were highly significant (***P < 0.001). E. C57BL/6 MEFs were induced with IFNγ and infected with T. gondii RH-YFP strain. Mean fluorescence intensities of Irga6 and Irgb6 were measured for selected PVs expressing no detectable (open circles), weak (grey filled circles) or strong Irga6 staining (black filled circles). The fluorescent intensity profiles of five representative PVs per group are displayed in Fig. S6. F. Photomicrograph of an IFNγ-stimulated MEF shown 2 h after double infection with ME49 strain (indicated by arrowhead) and RH-YFP strain T. gondii (green, indicated by arrow). The ME49 strain parasite shows intense Irgb6 (serum A20, red) accumulation at the PV while the RH-YFP in the same cell has no Irgb6 on the PV. G. IFNγ-stimulated MEFs were infected with T. gondii ME49 strain alone or simultaneously with ME49 and RH-YFP strains. Irgb6 (detected by serum A20) and Irga6 (detected by mAb 10D7) fluorescence intensities were measured on at least 30 ME49 PVs in singly and doubly infected cells. ME49 and RH-YFP were discriminated by the fluorescent signal from RH-YFP. The arithmetic means are given as horizontal lines. The loading of Irga6 and Irgb6 onto PVs of avirulent ME49 strain T. gondii was unaffected by the simultaneous presence of virulent RH-YFP.
Mentions: Reduced loading of Irgb6 on virulent type I strain RH PVs has recently been reported in MEFs and macrophages, correlating with reduced vacuolar disruption (Zhao et al., 2009a,b,c;). The data shown above were all based on infection of cells with the avirulent T. gondii strain, ME49. As shown in Fig. 8A, two type I strains, BK and RH, gave grossly defective loading of Irgb6, while two avirulent type II strains, ME49 and NTE, and an avirulent type III strain, CTG, showed the familiar high frequency of Irgb6-loaded vacuoles. Loading of all IRG proteins was attenuated on RH strain vacuoles (Fig. 8B) but two different effects were apparent. Irgb6 and Irgb10 loaded onto very few vacuoles, while for Irga6 or Irgd the number of loaded vacuoles was significantly but not dramatically reduced but the amount loaded per vacuole was generally much lower, documented for Irga6 in Figs 8C and D and S6. The few vacuoles loaded with Irgb6 were loaded very heavily (Fig. 8C and D) and were additionally all intensely loaded for Irga6 (Figs 8E and S6) and Irgd (J.P. Hunn and S. Könen-Waisman, unpublished data). In addition to their implications for the nature of virulence, these results also support the concept that Irgb6 and Irgb10 function as loading pioneers. In their absence, the remaining IRG proteins have difficulty gaining a foothold on the vacuole. In IFN-induced MEFs co-infected with RH-YFP and unlabelled ME49, ME49-containing vacuoles could be intensely coated with Irgb6 while RH-containing vacuoles in the same cell had none (Fig. 8F), and the loading intensity of Irgb6 and Irga6 on ME49 PVs was unaffected by the presence of RH-YFP vacuoles (Fig. 8G). It is therefore unlikely that a diffusible molecule from the virulent strain is responsible for reduced Irgb6 loading. Our results and conclusions confirm those of Zhao et al. (Zhao et al., 2009c).

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