Abstract
Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.
Highlights
Irgb[6] and Irgb[10] were immunoprecipitated from IFNγ-induced or transiently transfected, metabolically labelled L929 cells and infected with virulent T. gondii strain RH-YFP or CTG transgenic T. gondii strains. (A) A weak 32P-inorganic phosphate labelled band corresponding to Irgb[6] was immunoprecipitated with mouse monoclonal anti-Irgb[6] antibody, B34, only from IFNγ-induced cells
A very weakly labelled nonspecific band running above the positive control Irga[6] protein is indicated by an asterisk. (B) A montage of autoradiograms showing immunoprecipitation of Irga[6], Irgb[10] from RH-YFP-infected, CTG transgenic strain-infected, or uninfected L929 cells labelled with 33P-phosphoric acid
Phosphorylated Irgb[10] could be reproducibly immunoprecipitated from uninfected, IFNγ-induced cells, but the signal was enhanced by RH-YFP infection
Summary
S6 Fig. Irgb[6] and Irgb[10] are phosphorylated upon type I virulent strain infection. Irgb[6] and Irgb[10] were immunoprecipitated from IFNγ-induced or transiently transfected, metabolically labelled L929 cells and infected with virulent T. gondii strain RH-YFP or CTG transgenic T. gondii strains.
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