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Differential impacts on host transcription by ROP and GRA effectors from the intracellular parasite Toxoplasma gondii

ABSTRACT: The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the hostâ€™s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.

ORGANISM(S): Toxoplasma gondii RH

PROVIDER: GSE145800 | GEO | 2020/02/25

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.

Project description:Toxoplasma gondii is an obligate intracellular parasite that must establish a favorable environment in the host cells in which it replicates. To do this, it controls key host cell processes by MYR-dependent translocation of dense granule proteins from the parasitophorous vacuole into the host cytosol. Using RNASeq, we previously reported that mutants lacking the MYR translocation apparatus fail to elicit a key set of host responses related to regulation of the cell cycle, including cyclin E and other targets of E2F transcription factors. As no previously reported effector was known to mediate such an effect, we report here our application of a set of criteria to proteins secreted by Toxoplasma leading to our discovery of a novel effector protein that induces the host to produce cyclin E. This inducer of Host Cyclin E (HCE1) is exported from the parasitophorous vacuole in a manner dependent on MYR1 and ASP5, another component of the export system, where it then localizes to the host’s nucleus. HCE1 expression is important for parasite growth as those lacking it produce plaques that are ~35% smaller in area than wild type parasites. RNASeq of host cells infected with parasites lacking HCE1 compared to those infected with wild type parasites shows a complete loss of the parasite’s ability to modulate key host cell cycle genes including those related to the E2F axis. Immunoprecipitation of HCE1 from infected host cells shows that HCE1 efficiently binds elements of the cyclin E regulatory complex: DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, which is not otherwise capable of up-regulating cyclin E, or in uninfected HFFs, shows nuclear localization of the expressed protein and results in strong cyclin E up-regulation. Thus, HCE1 is a novel, stand-alone effector protein that is necessary and sufficient to impact the E2F-axis of transcription resulting in co-opting of host functions to Toxoplasma’s advantage.

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Differential impacts on host transcription by ROP and GRA effectors from the intracellular parasite Toxoplasma gondii

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