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Translocation of dense granule effectors across the parasitophorous vacuole membrane in Toxoplasma-infected cells requires the activity of ROP17, a rhoptry protein kinase

ABSTRACT: 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 Δrop17 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE133144 | GEO | 2019/07/02

REPOSITORIES: GEO

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Project description:The intracellular parasite Toxoplasma gondii uses a diverse repertoire of effector molecules to manipulate its host cell. The first effectors to be introduced into the host cell derive from rhoptries (ROPs) and are secreted exclusively during invasion. Once the parasite has established a parasitophorous vacuole, a subset of secreted dense granule effector proteins (GRAs) are then translocated across the parasitophorous vacuolar membrane in a MYR1-dependent manner to enter the host cell cytoplasm and nucleus. Translocation of these GRA effectors is also dependent on the activity of Toxoplasma Aspartyl protease 5 (ASP5) which either directly cleaves the effectors or potentiates translocation in some other way. To determine the totality of MYR1-dependent (predominantly GRA) and MYR1-independent (e.g., ROP) host responses as well as the degree of overlap between MYR1- and ASP5-dependent effectors, we analyzed the RNAseq expression profile of human foreskin fibroblasts infected with wild type (RH strain) Toxoplasma gondii tachyzoites, RHΔmyr1, RHΔasp5 and their respective complements. We show that a majority subset of the differentially regulated host gene responses during infection with wild type parasites are modulated in a MYR1-dependent manner. Gene set enrichment analysis of the MYR1-dependent host responses indicates that, among others, there is a clear up-regulation related to E2F transcription factors and the cell cycle and a clear down-regulation in expression related to interferon signaling. The ASP5-dependent host gene responses were less numerous and encompassed fewer gene sets than the MYR1-dependent responses, indicating that translocation and/or activity of a subset of effectors is MYR1- but not ASP5-dependent. Consistent with this, we show that even though MYR1 is normally cleaved by ASP5, such cleavage is not necessary for MYR1’s role in translocation. Interestingly, some responses were seen upon infection with RHΔmyr1 but not RH-WT; these previously “hidden” responses are likely due to the normally counter-balancing action of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1-dependent provide new insight into the relative contribution of GRA- vs. ROP-mediated effectors in co-opting host cell functions.

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Translocation of dense granule effectors across the parasitophorous vacuole membrane in Toxoplasma-infected cells requires the activity of ROP17, a rhoptry protein kinase

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