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Background: The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane. Results: Irga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRalpha). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism. Conclusions: The catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins ex

Toxoplasma gondii is a widespread protozoan parasite infecting all warm-blooded animals and causing disease in immunocompromised individuals and in utero. The pathogen depends on the intracellular life style residing in a specialized organelle, termed parasitophorous vacuole (PV), in order to survive and replicate. Cell-autonomous immunity, regulated by IFNg, is essential to restrict growth of the parasite in mouse and man. In mouse cells, resistance to T. gondii is mediated by the family of IFN-inducible IRG proteins (p47 GTPases). Upon infection several IRG proteins associate with the PV and participate in vesiculation of the PV membrane leading to demise of the parasite and necrotic death of the cell. Until now, despite intrinsic interest, the phenomenon of IRG protein loading onto PV has not been well studied. In human cells, depletion of cellular tryptophan by IDO has been reported as the major mechanism of restriction of T. gondii proliferation exerted by IFNg. IDO-independent restriction of T. gondii growth has been reported but not followed up. The process of IRG protein association with T. gondii vacuoles emerged as a rapid, organized and diffusion-driven event where multiple resistance proteins sequentially bind to the vacuolar membrane forming homomeric and heteromeric complexes. The efficient loading process requires the autophagy factor Atg5 regulating correct localisation of IRG proteins prior to infection. Virulent strains of T. gondii inhibit IRG protein association with PVs independently of individual virulence determinants ROP5, ROP16 and ROP18. Impaired loading of IRG proteins onto T. gondii vacuoles leads to reduced elimination of the parasite in IFNg-stimulated cells, underlining the importance of the phenomenon in cell-autonomous immunity to T. gondii. This study shows that density of cultured cells is the key factor in determining the mode of T. gondii control in primary human cells. IFNg-induced, proliferating cells control parasite replicati

Toxoplasma gondii is a widespread protozoan parasite infecting all warm-blooded animals and causing disease in immunocompromised individuals and in utero. The pathogen depends on the intracellular life style residing in a specialized organelle, termed parasitophorous vacuole (PV), in order to survive and replicate. Cell-autonomous immunity, regulated by IFNg, is essential to restrict growth of the parasite in mouse and man. In mouse cells, resistance to T. gondii is mediated by the family of IFN-inducible IRG proteins (p47 GTPases). Upon infection several IRG proteins associate with the PV and participate in vesiculation of the PV membrane leading to demise of the parasite and necrotic death of the cell. Until now, despite intrinsic interest, the phenomenon of IRG protein loading onto PV has not been well studied. In human cells, depletion of cellular tryptophan by IDO has been reported as the major mechanism of restriction of T. gondii proliferation exerted by IFNg. IDO-independent restriction of T. gondii growth has been reported but not followed up. The process of IRG protein association with T. gondii vacuoles emerged as a rapid, organized and diffusion-driven event where multiple resistance proteins sequentially bind to the vacuolar membrane forming homomeric and heteromeric complexes. The efficient loading process requires the autophagy factor Atg5 regulating correct localisation of IRG proteins prior to infection. Virulent strains of T. gondii inhibit IRG protein association with PVs independently of individual virulence determinants ROP5, ROP16 and ROP18. Impaired loading of IRG proteins onto T. gondii vacuoles leads to reduced elimination of the parasite in IFNg-stimulated cells, underlining the importance of the phenomenon in cell-autonomous immunity to T. gondii. This study shows that density of cultured cells is the key factor in determining the mode of T. gondii control in primary human cells. IFNg-induced, proliferating cells control parasite replicati