Role of the Secreted Kinase ROP18 in Toxoplasma gondii Pathogenesis

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular parasite that must establish a parasitophorous vacuole to serve as the replication-permissive niche during infection. In order to set up this safe haven, the parasite uses directed protein secretion from specialized organelles called rhoptries to modify the vacuolar membrane. Forward genetic analysis identifies a serine-threonine protein kinase, ROP18, localizing to the rhoptry compartment as a major virulence determinant of the highly pathogenic type I strains of Toxoplasma gondii. Kinase activity of ROP18 is critical to pathogenesis, as point mutations disrupting catalytic activity fail to confer parasite virulence. However, the mechanism by which ROP18 enhances virulence, the regulation of ROP18 catalytic activity, and the function of vacuolar membrane association were not known. Following secretion, tethering of ROP18 to the cytoplasmic face of the vacuolar membrane was found to be mediated by arginine-rich, amphipathic alpha helices proximal to the kinase domain. Mutations in these helices did not affect secretion of the kinase from rhoptries during parasite invasion but dissociated ROP18 from the vacuolar membrane and rendered the parasite avirulent, demonstrating that proper localization of the kinase in infected cells is critical for its function. The parasite-modified vacuolar membrane provides an interface on which Toxoplasma can directly interact with the host to alter infection outcomes and, conversely, provides a foreign surface for the host to attack. In response to infection, the host up-regulates a family of immunity-related GTPases (IRGs) in the dynamin superfamily that accumulates on the parasite vacuole, resulting in vesiculation and rupture of the vacuolar membrane and death of the parasite following dissolution of its safe haven. Cellular studies revealed the catalytic activity of ROP18 was both necessary and sufficient to subvert the IRG pathway by preventing the accumulation of IRGs on the vacuolar membrane and thus enhanced parasite survival in a kinase-dependent manner. To ascertain the kinase substrate mediating IRG inhibition, biochemical and mass spectrometry analyses were employed establishing the regulatory switch region I in the GTPase domain of IRGs as the target of ROP18 phosphorylation, hence providing mechanistic evidence for the virulence-enhancing activity of ROP18. Genetic evidence from natural Toxoplasma isolates suggested ROP18 and the predicted pseudokinase ROP5 might converge on the same pathway controlling pathogenesis. Analysis of ROP18- and ROP5-deficient strains demonstrated a requirement for ROP5 in blocking the IRG pathway and revealed ROP18 kinase activity is greatly diminished in the absence of ROP5 expression. Collectively, these results demonstrate that Toxoplasma deploys the active kinase ROP18 to the vacuolar membrane to offset the host immune response and this activity is regulated by the secreted pseudokinase ROP5