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Ribo-seq of Toxoplasma-infected host cells

ABSTRACT: Toxoplasma gondii is a ubiquitous obligate intracellular parasite that infects the nucleated cells of warm-blooded animals. From within the parasitophorous vacuole in which they reside, Toxoplasma tachyzoites secrete an arsenal of effector proteins that can reprogram host gene expression to facilitate parasite survival and replication. Gaining a better understanding of how host gene expression is altered upon infection is central for understanding parasite strategies for host invasion and for developing new parasite therapies. Here, we applied ribosome profiling coupled with mRNA measurements to concurrently study gene expression in the parasite and in host human foreskin fibroblasts. By examining the parasite transcriptome and translatome, we identified potential upstream open reading frames that may permit the stress-induced preferential translation of parasite mRNAs. We also determined that tachyzoites reduce host death-associated pathways and increase survival, proliferation, and motility in both quiescent and proliferative host cell models of infection. Additionally, proliferative cells alter their gene expression in ways consistent with massive transcriptional rewiring while quiescent cells were best characterized by re-entry into the cell cycle. We also identified a translational control regimen consistent with mTOR activation in quiescent cells, and to a lesser degree in proliferative cells. This study illustrates the utility of the method for dissection of gene expression programs simultaneously in parasite and host.

ORGANISM(S): Toxoplasma gondii RH Homo sapiens

PROVIDER: GSE129869 | GEO | 2019/05/17

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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Ribo-seq of Toxoplasma-infected host cells

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