GSE133144
Translocation of dense granule effectors across the parasitophorous vacuole membrane in Toxoplasma-infected cells requires the activity of ROP17, a rhoptry protein kinase
Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors.  The machinery that translocates these GRA effectors has recently been partially elucidated, revealing 3 components, MYR1, MYR2 and MYR3. To determine if other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one that has a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Whole-genome sequencing showed that this mutant indeed has a missense mutation in a gene encoding a known serine/threonine protein kinase, ROP17, which has not previously been reported to be involved in GRA translocation. ROP17 resides at the PVM in infected cells and has previously been known for its activity in phosphorylating and, thereby, inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM, and that translocation across the PVM surrounding a ?rop17 mutant can be rescued “in trans” by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues for a role for ROP17 at the PVM, not within the parasites or parasitophorous vacuole space. Hence, ROP17 plays a crucial part in GRA translocation, adding another function to the role it plays in modulating the interaction of Toxoplasma tachyzoites and the infected host cell. Overall design: RNASeq of human foreskin fibroblasts, infected with Toxoplasma, harvested 6 hours post infection. Samples are: Mock infected, infected with a WT RH, RH?myr1,  RH?rop17