Toxoplasma protein phosphatase PPKL phosphoproteomics data
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Toxoplasma Gondii (ncbitaxon:5811)
SUBMITTER: 
Emma Doud Gustavo Arrizabalaga
PROVIDER: MSV000092849 | MassIVE |
SECONDARY ACCESSION(S): PXD045354
REPOSITORIES: MassIVE
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Project description:Toxoplasma protein phosphatase PPKL TurboID MS data of the paper "The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii"
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Project description:Toxoplasma protein phosphatase PPKL Co-IP MS data of the paper "The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii".
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Project description:Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.
2023-11-28 | PXD041907 | Pride
Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite. RNA samples were isolated in triplicates from RH-hxgprt parent strain (W), PRMT1 knockout (K) strain and PRMT1 knockout strain complemented with RFP-tagged PRMT1 protein (C). Parasites were grown for 32h at 37C. Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies).
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Project description:Toxoplasma gondii is a globally distributed obligate intracellular parasite which can cause zoonotic toxoplasmosis with great harms. The average death time of mice that infected with Toxoplasma gondii RH strain tachyzoites recovered from the liquid nitrogen was shortened after multiple generations. It has been reported that the parasite is in a state of static virulence during cryopreservation and the virulence of the protozoan parasite can be enhanced after continuous passages in hosts under laboratory conditions. However, no research has been conducted to elucidate its biological mechanism. Herein, we sequenced the T. gondii transcriptome using RNA-Seq technology and performed de novo assembly to investigated the virulence factors expression changes by comparing gene expression profiles between incipiently recovered and completely resuscitated tachyzoites. Transcriptome analysis identified 1,951 differentially expressed transcripts in infected liver, of which 1,752 were significantly downregulated and 199 upregulated. We identified many differentially expressed proteins and genes, including serine/threonine kinase, calnexin, myosin and microtubule-associated protein which have previously been reported to be either involved in cell adhesion, parasite gliding or participate in cell invasion. The great majority of the virulence factors including microneme proteins, rhoptry proteins and dense granule proteins were upregulated in fully recovered tachyzoites. The enhanced virulence of recovered Toxoplasma gondii RH strain from the liquid nitrogen is associated with the up-regulated expression of MICs, ROPs and GRAs. Our data will facilitate future genomic research and in-depth annotation of Toxoplasma gondii RH strain genomes. This study provides a profile of the candidate genes that are suspected to be involved with virulence enhancement of recovered Toxoplasma gondii RH strain tachyzoites. Many further studies should be carried out to confirm the function of the candidate genes. Moreover, the preliminary identification of genes and pathways exhibiting differential expression in complete resuscitation stage may further our general understanding of virulence enhancement in this parasite.
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Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite.
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Project description:Phenotypic switching from tachyzoite to bradyzoite and vice versa is the fundamental mechanism underpinning the pathogenicity and adaptability of the protozoan parasite Toxoplasma gondii. Accumulation of cytoplasmic starch granules is a hallmark of the quiescent bradyzoite stage. The regulatory factors and mechanisms that contribute to amylopectin storage in bradyzoites remain incompletely known. Here, we show that T. gondii protein phosphatase 2A (PP2A) holoenzyme is composed of a catalytic subunit (PP2A-C), a structural subunit (PP2A-A) and a regulatory subunit (PP2A-B). Disruption of any of these subunits increased starch accumulation and disrupted the parasite differentiation. The putative PP2A holoenzyme substrates were identified by phosphoproteomics. PP2A contributes to the regulation of amylopectin metabolism via dephosphorylation of calcium-dependent protein kinase 2 at S679. Several putative PP2A substrates were found to play important roles in bradyzoite differentiation. Our findings establish PP2A as an integral component of the regulatory network mediating amylopectin metabolism and tachyzoite-bradyzoite transformation in T. gondii.
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Project description:Toxoplasma gondii multiplies inside a parasitophorous vacuole in the host cell. Several parasite proteins have been described that hijack host signaling pathways, which mostly originate from the rhoptry organelles. We report here the identification and characterization of GRA16, the first dense granule protein shown to be exported through the parasitophorous vacuole membrane and to reach the host cell nucleus. Transcriptomic analysis revealed that GRA16 positively modulates the expression of host genes involved in cell-cycle progression and the p53 tumor suppressor pathway. We show that GRA16 directly binds two host enzymes, the deubiquitinase HAUSP and the phosphatase PP2A, and that GRA16 alters p53 protein levels in a HAUSP-dependent manner and induces the nuclear translocation of the PP2A holoenzyme. Therefore GRA16 is a novel regulator of the HAUSP/p53 pathway and together with GRA15, emerge as a subfamily of new dense granule proteins exported beyond the tachyzoites-hosting vacuole to subvert the host transcriptome. Mouse bone marrow-derived macrophages (BMDM) or Human foreskin fibroblasts (HFFs) were infected with the following Toxoplasma gondii strains: - RHku80 WT versus RHku80(deltaGRA16) mutant (in BMDM) - Pruku80 WT versus Pruku80(deltaGRA16) mutant (in BMDM) - RHku80 WT versus RHku80(deltaGRA16) mutant (in HFF)
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Project description:To identify accessible chromatin regions in the human host cells during Toxoplasma parasite infection (uninfected, RH-infected and Pru-infected human foreskin fibroblasts) and in the obligate intracellular parasite Toxoplasma gondii (Type 1 RH strain and Type 2 Pru strain), ATAC-seq was performed.
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Project description:The lytic cycle of the protozoan parasite Toxoplasma gondii, which involves a brief sojourn in the extracellular space, is characterized by defined transcriptional profiles. For an obligate intracellular parasite that is shielded from the cytosolic host immune factors by a parasitophorous vacuole, the brief entry into the extracellular space is likely to exert enormous stress. Due to its role in cellular stress response, we hypothesize that translational control plays an important role in regulating gene expression in Toxoplasma during the lytic cycle. Unlike transcriptional profiles, insights into genome-wide translational profiles of Toxoplasma gondii are lacking. We have performed genome-wide ribosome profiling, coupled with high throughput RNA sequencing, in intracellular and extracellular Toxoplasma gondii parasites to investigate translational control during the lytic cycle. Results: Although differences in transcript abundance were mostly mirrored at the translational level, we observed significant differences in the abundance of ribosome footprints between the two parasite stages. Furthermore, our data suggest that mRNA translation in the parasite is potentially regulated by mRNA secondary structure and upstream open reading frames.
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