Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii

Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6-specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e. g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity-related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e. g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6-specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18-specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors

Molecular mechanism for the control of virulent Toxoplasma gondii infections in wild-derived mice

Some strains of the protozoan parasite Toxoplasma gondii (such as RH) are virulent in laboratory mice because they are not restricted by the Immunity-Related GTPase (IRG) resistance system in these mouse strains. In some wild-derived Eurasian mice (such as CIM) on the other hand, polymorphic IRG proteins inhibit the replication of such virulent T. gondii strains. Here we show that this resistance is due to direct binding of the IRG protein Irgb2-b1(CIM) to the T. gondii virulence effector ROP5 isoform B. The Irgb2-b1 interface of this interaction is highly polymorphic and under positive selection. South American T. gondii strains are virulent even in wild-derived Eurasian mice. We were able to demonstrate that this difference in virulence is due to polymorphic ROP5 isoforms that are not targeted by Irgb2-b1(CIM), indicating co-adaptation of host cell resistance GTPases and T. gondii virulence effectors