The obligate intracellular parasite Toxoplasma gondii secretes effector proteins into the host cell that manipulate the immune response allowing it to establish a chronic infection. Crosses between the types I, II and III strains, which are prevalent in North America and Europe, have identified several secreted effectors that determine strain differences in mouse virulence. The polymorphic rhoptry protein kinase ROP18 was recently shown to determine the difference in virulence between type I and III strains by phosphorylating and inactivating the interferon-γ (IFNγ)-induced immunity-related GTPases (IRGs) that promote killing by disrupting the parasitophorous vacuole membrane (PVM) in murine cells. The polymorphic pseudokinase ROP5 determines strain differences in virulence through an unknown mechanism. Here we report that ROP18 can only inhibit accumulation of the IRGs on the PVM of strains that also express virulent ROP5 alleles. In contrast, specific ROP5 alleles can reduce IRG coating even in the absence of ROP18 expression and can directly interact with one or more IRGs. We further show that the allelic combination of ROP18 and ROP5 also determines IRG evasion and virulence of strains belonging to other lineages besides types I, II and III. However, neither ROP18 nor ROP5 markedly affect survival in IFNγ-activated human cells, which lack the multitude of IRGs present in murine cells. These findings suggest that ROP18 and ROP5 have specifically evolved to block the IRGs and are unlikely to have effects in species that do not have the IRG system, such as humans.
Toxoplasma gondii can infect any warm-blooded animal and is transmitted orally by consumption of tissue cysts. To facilitate transmission, the parasite must balance induction and evasion of host immune responses to allow parasite growth and persistence, while avoiding excessive parasite burden, which can kill the host before infectious cysts are formed. Different strains of Toxoplasma have likely evolved specific effector molecules to modulate the immune responses of different hosts. In many mammals, including mice but not humans, the cytokine interferon gamma (IFNγ) induces the immunity-related GTPases (IRGs), which are essential to the murine immune response to Toxoplasma. They function by binding to and disrupting the parasite-containing vacuole. However, some Toxoplasma strains prevent the IRGs from disrupting the parasitophorous vacuole. It was previously shown that the secreted Toxoplasma kinase ROP18 promotes virulence in mice by phosphorylating the IRGs, leading to their inactivation. We report that ROP18 requires another virulence factor, the secreted pseudokinase ROP5, to prevent IRG accumulation, and these two proteins determine the majority of strain differences in IRG evasion, even for divergent strains for which virulence determinants have not been studied. Additionally, we show that ROP18 and ROP5 do not affect Toxoplasma survival in IFNγ-stimulated human cells. Thus, ROP18 and ROP5 are strain- and host-specific determinants of Toxoplasma immune evasion.