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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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Loading of Irga6 at the T. gondii ME49 strain PV is enhanced by the presence of other IRG proteins of the GKS group. gs3T3-Irga6 cells were induced with IFNγ or Mifepristone. At the same time, Mifepristone-induced cells were transfected with pools of constructs (see Experimental procedures for experimental details) expressing either the three GMS proteins, (Irgm1, Irgm2 and Irgm3) alone to permit access of Irga6 to the PV (3GMS) or, in addition to the 3GMS proteins, also Irgb6 (3GMS + b6), Irgd (3GMS + d) or both Irgb6 and Irgd (5IRGs). After 24 h, cells were infected with T. gondii ME49 strain for 2 h. Irga6 was detected at the PV in transfected cells using mAb 10E7 in immunofluorescence. Transfected cells were identified by staining for Irgm2 with the H53/3 serum. The arithmetic means are given as horizontal lines. Vacuolar loading of Irga6 was significantly enhanced by addition of Irgb6 or Irgd. The P-values are given on the figure (***P < 0.001).
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fig07: Loading of Irga6 at the T. gondii ME49 strain PV is enhanced by the presence of other IRG proteins of the GKS group. gs3T3-Irga6 cells were induced with IFNγ or Mifepristone. At the same time, Mifepristone-induced cells were transfected with pools of constructs (see Experimental procedures for experimental details) expressing either the three GMS proteins, (Irgm1, Irgm2 and Irgm3) alone to permit access of Irga6 to the PV (3GMS) or, in addition to the 3GMS proteins, also Irgb6 (3GMS + b6), Irgd (3GMS + d) or both Irgb6 and Irgd (5IRGs). After 24 h, cells were infected with T. gondii ME49 strain for 2 h. Irga6 was detected at the PV in transfected cells using mAb 10E7 in immunofluorescence. Transfected cells were identified by staining for Irgm2 with the H53/3 serum. The arithmetic means are given as horizontal lines. Vacuolar loading of Irga6 was significantly enhanced by addition of Irgb6 or Irgd. The P-values are given on the figure (***P < 0.001).

Mentions: The three GMS proteins, Irgm1, Irgm2 and Irgm3, characterized by a unique substitution of methionine for lysine in the G1 motif of the nucleotide-binding site (Boehm et al., 1998), control the GTPase cycle of the conventional GKS proteins, Irga6, Irgb6 and Irgd (Hunn et al., 2008). In the absence of the GMS proteins, Irga6 and Irgb6 form nucleotide-dependent cytoplasmic aggregates that are probably caused by premature GTP binding and activation (Hunn et al., 2008; Papic et al., 2008). Irga6, expressed alone in 3T3 cells from a Mifepristone-inducible construct, was unable to relocate to the PVM of infecting T. gondii strain, but vacuolar location was restored if the three GMS proteins were also introduced by transfection (Hunn et al., 2008). However the frequency of Irga6-loaded vacuoles was low (Hunn et al., 2008) and accumulation of Irga6 at the PVM was weak (Fig. 7 and J.P. Hunn, unpublished data). The co-ordinated loading described above hinted that Irgb6 (and/or other GKS proteins) might be necessary for complete loading of Irga6. We therefore reconstituted Mifepristone-induced gs3T3-Irga6 fibroblasts with constructs expressing Irgb6, Irgd or both as well as the three GMS proteins and determined the Irga6 signal intensity on T. gondii PVM 2 h after infection. Coexpression of either Irgb6 or Irgd or both caused a significant increase in the Irga6 signal at the PVM without increasing the frequency of Irga6-loaded vacuoles (Fig. 7, Hunn et al., 2008 and J.P. Hunn, unpublished data). Thus other IRG proteins of the GKS group are required for efficient Irga6 loading of the PVM. Unlike Irga6, Irgd does not load at all on to the PVM if only the three GMS proteins are also present, but loads if Irgb6 is also present (J.P. Hunn, unpublished data). Thus the loading of the PVM by GKS proteins is a highly cooperative process, with Irgb6 and probably also Irgb10 arriving as pioneers and subsequently becoming stabilized by the arrival of Irga6 and Irgd. In this context, it may be significant that both Irgb6 and Irgb10 can load, albeit inefficiently, onto T. gondii vacuoles in the absence of other IRG proteins (Hunn et al., 2008, Fig. 7 and J.P. Hunn, unpublished data). A different explanation for the presence of Irgm2 and Irgm3 at the vacuole is presented in the Discussion.


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)

Loading of Irga6 at the T. gondii ME49 strain PV is enhanced by the presence of other IRG proteins of the GKS group. gs3T3-Irga6 cells were induced with IFNγ or Mifepristone. At the same time, Mifepristone-induced cells were transfected with pools of constructs (see Experimental procedures for experimental details) expressing either the three GMS proteins, (Irgm1, Irgm2 and Irgm3) alone to permit access of Irga6 to the PV (3GMS) or, in addition to the 3GMS proteins, also Irgb6 (3GMS + b6), Irgd (3GMS + d) or both Irgb6 and Irgd (5IRGs). After 24 h, cells were infected with T. gondii ME49 strain for 2 h. Irga6 was detected at the PV in transfected cells using mAb 10E7 in immunofluorescence. Transfected cells were identified by staining for Irgm2 with the H53/3 serum. The arithmetic means are given as horizontal lines. Vacuolar loading of Irga6 was significantly enhanced by addition of Irgb6 or Irgd. The P-values are given on the figure (***P < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig07: Loading of Irga6 at the T. gondii ME49 strain PV is enhanced by the presence of other IRG proteins of the GKS group. gs3T3-Irga6 cells were induced with IFNγ or Mifepristone. At the same time, Mifepristone-induced cells were transfected with pools of constructs (see Experimental procedures for experimental details) expressing either the three GMS proteins, (Irgm1, Irgm2 and Irgm3) alone to permit access of Irga6 to the PV (3GMS) or, in addition to the 3GMS proteins, also Irgb6 (3GMS + b6), Irgd (3GMS + d) or both Irgb6 and Irgd (5IRGs). After 24 h, cells were infected with T. gondii ME49 strain for 2 h. Irga6 was detected at the PV in transfected cells using mAb 10E7 in immunofluorescence. Transfected cells were identified by staining for Irgm2 with the H53/3 serum. The arithmetic means are given as horizontal lines. Vacuolar loading of Irga6 was significantly enhanced by addition of Irgb6 or Irgd. The P-values are given on the figure (***P < 0.001).
Mentions: The three GMS proteins, Irgm1, Irgm2 and Irgm3, characterized by a unique substitution of methionine for lysine in the G1 motif of the nucleotide-binding site (Boehm et al., 1998), control the GTPase cycle of the conventional GKS proteins, Irga6, Irgb6 and Irgd (Hunn et al., 2008). In the absence of the GMS proteins, Irga6 and Irgb6 form nucleotide-dependent cytoplasmic aggregates that are probably caused by premature GTP binding and activation (Hunn et al., 2008; Papic et al., 2008). Irga6, expressed alone in 3T3 cells from a Mifepristone-inducible construct, was unable to relocate to the PVM of infecting T. gondii strain, but vacuolar location was restored if the three GMS proteins were also introduced by transfection (Hunn et al., 2008). However the frequency of Irga6-loaded vacuoles was low (Hunn et al., 2008) and accumulation of Irga6 at the PVM was weak (Fig. 7 and J.P. Hunn, unpublished data). The co-ordinated loading described above hinted that Irgb6 (and/or other GKS proteins) might be necessary for complete loading of Irga6. We therefore reconstituted Mifepristone-induced gs3T3-Irga6 fibroblasts with constructs expressing Irgb6, Irgd or both as well as the three GMS proteins and determined the Irga6 signal intensity on T. gondii PVM 2 h after infection. Coexpression of either Irgb6 or Irgd or both caused a significant increase in the Irga6 signal at the PVM without increasing the frequency of Irga6-loaded vacuoles (Fig. 7, Hunn et al., 2008 and J.P. Hunn, unpublished data). Thus other IRG proteins of the GKS group are required for efficient Irga6 loading of the PVM. Unlike Irga6, Irgd does not load at all on to the PVM if only the three GMS proteins are also present, but loads if Irgb6 is also present (J.P. Hunn, unpublished data). Thus the loading of the PVM by GKS proteins is a highly cooperative process, with Irgb6 and probably also Irgb10 arriving as pioneers and subsequently becoming stabilized by the arrival of Irga6 and Irgd. In this context, it may be significant that both Irgb6 and Irgb10 can load, albeit inefficiently, onto T. gondii vacuoles in the absence of other IRG proteins (Hunn et al., 2008, Fig. 7 and J.P. Hunn, unpublished data). A different explanation for the presence of Irgm2 and Irgm3 at the vacuole is presented in the Discussion.

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