Secretory polymorphic serine/threonine kinases control pathogenesis of Toxoplasma gondii in the mouse. Genetic studies show that the pseudokinase ROP5 is essential for acute virulence, but do not reveal its mechanism of action. Here we demonstrate that ROP5 controls virulence by blocking IFN-γ mediated clearance in activated macrophages. ROP5 was required for the catalytic activity of the active S/T kinase ROP18, which phosphorylates host immunity related GTPases (IRGs) and protects the parasite from clearance. ROP5 directly regulated activity of ROP18 in vitro, and both proteins were necessary to avoid IRG recruitment and clearance in macrophages. Clearance of both the Δ rop5 and Δ rop18 mutants was reversed in macrophages lacking Irgm3, which is required for IRG function, and the virulence defect was fully restored in Irgm3 −/− mice. Our findings establish that the pseudokinase ROP5 controls the activity of ROP18, thereby blocking IRG mediated clearance in macrophages. Additionally, ROP5 has other functions that are also Irgm3 and IFN-γ dependent, indicting it plays a general role in governing virulence factors that block immunity.
The ability of microorganisms to cause disease in their hosts is often mediated by proteins that are secreted by the pathogen into the host cell as a means of disarming host signaling. Previous studies with the protozoan parasite Toxoplasma gondii have revealed that secretion of parasite protein kinases into the host cell mediates virulence in mouse, a natural host for transmission. Curiously, some of these virulence factors are active protein kinases, while other related pseudokinases lack enzymatic activity; hence, it was unclear how they functioned in promoting virulence. In the present work we demonstrate that ROP5, an inactive member of this protein kinase family, regulates the active protein kinase ROP18, which normally prevents clearance of the parasite in interferon-activated macrophages. Allosteric regulation of enzymes is a common theme in biology, but this is the first example of such a mechanism regulating a pathogen virulence factor. The potential advantage of such a layered process is that it might allow greater temporal or spatial control and perhaps protect the parasite from disabling strategies by the host.