A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice

Young, Joanna C.; Dominicus, Caia; Wagener, Jeanette; Butterworth, Simon; Ye, Xingda; Kelly, Gavin; Ordan, Merav; Saunders, Becky; Instrell, Rachael; Howell, Michael; Stewart, Aengus; Treeck, Moritz

doi:10.1038/s41467-019-11855-w

Cited by 64 publications

(97 citation statements)

References 44 publications

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“…It could be that the proteins have a subtle effect on growth that we have not been able to measure or that they are important in another host species, parasite stage, or genetic background that we have not tested here. However, neither SFP1 nor GRA29 showed a reduction in fitness in vivo (33) in our recent more sensitive in vivo CRISPR screen, supporting a nonessential role under these conditions.…”

Section: Discussionmentioning

confidence: 74%

Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

Young

Broncel

Teague

et al. 2020

mSphere

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The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion. IMPORTANCE Toxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

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Section: Discussionmentioning

confidence: 74%

Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

Young

Broncel

Teague

et al. 2020

mSphere

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“…Further study is warranted to determine this synergistic or additive effect. Reassuringly, while this manuscript was under review, both Fox et al and Young et al showed that GRA12 contribute to parasites virulence in vivo and is dispensable for lytic cycle in vitro.…”

Section: Discussionmentioning

confidence: 90%

Novel roles of dense granule protein 12 (GRA12) inToxoplasma gondiiinfection

et al. 2020

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are contributed equally to this work. AbstractDense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii-host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity-related GTPase a6 (Irga6) onto the PV membrane.The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice. K E Y W O R D Sdense granule protein 12, immunity-related GTPases, intravacuolar network, rhoptry protein 18, Toxoplasma gondii, virulence 3166 | WANG et Al. SUPPORTING INFORMATIONAdditional supporting information may be found online in the Supporting Information section. How to cite this article: Wang J-L, Bai M-J, Elsheikha HM, et al. Novel roles of dense granule protein 12 (GRA12) in Toxoplasma gondii infection.

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“…These findings substantiate previous evidence that at least some MDGs suppress host responses induced by other parasite-driven transcriptomic perturbations ( 31 ). They may also explain the recent finding that the avirulent phenotype of Δ myr1 parasites during in vivo mouse infections is rescuable by coinfecting animals with both wild-type and MYR1-deficient parasites ( 46 ); parasites expressing MYR1 may induce host cells to secrete paracrine factors that suppress the transcription of inflammatory gene products that would otherwise limit Δ myr1 parasite infections. Note that while this article was in preparation, a transcriptomic study of U-I macrophages was reported by Chen and colleagues ( 44 ).…”

Section: Discussionmentioning

confidence: 98%

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

Rastogi

Xue

Quake

et al. 2020

mBio

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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 ultrapure 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 to 3 h postinfection 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 host’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 proinflammatory 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 suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes. IMPORTANCE This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

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A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice

Cited by 64 publications

References 44 publications

Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

Phosphorylation of Toxoplasma gondii Secreted Proteins during Acute and Chronic Stages of Infection

Novel roles of dense granule protein 12 (GRA12) inToxoplasma gondiiinfection

Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

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