MOSPD2-Toxoplasma Immunoprecipitation
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Toxoplasma Gondii Me49 Homo Sapiens (human)
TISSUE(S): Foreskin Fibroblast Cell Line
SUBMITTER: 
Abel Ferrel
LAB HEAD: John C Boothroyd
PROVIDER: PXD038158 | Pride | 2023-04-27
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 12 |
Similar Datasets
Project description: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.
2019-07-02 | GSE133144 | GEO
Project description:Cygan AM, Jean Beltran PM, Branon TC, Ting AY, Carr SA, Boothroyd JC. 2021.
Toxoplasma gondii is a ubiquitous, intracellular parasite that envelopes its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this crucial interface. Out of numerous host and parasite proteins with candidate PVM localization, we validate three novel parasite proteins (TGGT1_269950, TGGT1_215360, and TGGT1_217530) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as PVM-localized in infected human cells through immunofluorescent microscopy. These results significantly expand our knowledge of proteins present at the PVM and, given that three of the validated host proteins are components of the ESCRT machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface.
2021-02-10 | MSV000086833 | MassIVE
Project description: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 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 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.
2020-02-25 | GSE145800 | GEO
Project description:The apicomplexan parasite Toxoplasma gondii has a divergent mitochondrial electron transport chain (mETC). Succinate dehydrogenase (SDH), the second complex in the mETC and an enzyme in the TCA cycle, has 4 subunits in common model organisms, but Toxoplasma has a 9 subunit SDH complex. We performed immunoprecipitation of SDH, via an HA epitope tagged SDHB subunit, and the unrelated Complex III and analysed the elution via LFQ mass spectrometry, to confirm the composition of the divergent Toxoplasma gondii SDH complex.
2023-12-27 | PXD047027 | Pride
Project description:Toxoplasma gondii secretes various virulence effector molecules into host cells to disrupt host interferon-γ (IFN-γ)-dependent immunity. Among the effectors, ROP18 directly phosphorylates and inactivates IFN-inducible GTPases, such as immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs), leading to subversion of IFN-inducible GTPase-induced cell-autonomous immunity. The modes of action of ROP18 have been studied extensively; however, little is known about the molecular mechanism of how ROP18 is produced in the parasite itself. Here, we report a role of T. gondii transcription factor IWS1 in ROP18 mRNA expression in the parasite. Compared with wild-type virulent type I T. gondii, IWS1-deficient parasites showed dramatically increased loading of IRGs and GBPs onto the parasitophorous vacuole membrane (PVM). Moreover, IWS1-deficient parasites displayed decreased virulence in wild-type mice but retained normal virulence in mice lacking the IFN-γ receptor. Furthermore, IWS1-deficient parasites showed severely decreased ROP18 mRNA expression. Ectopic expression of ROP18 in IWS1-deficient parasites restored the decreased loading of effectors onto the PVM and in vivo virulence in wild-type mice. Taken together, these data demonstrate that T. gondii IWS1 regulates ROP18 mRNA expression to determine fitness in IFN-γ-activated host cells and mice.
2023-01-30 | GSE204926 | GEO
Project description:During their development within the vertebrate host, Plasmodium parasites infect hepatocytes and red blood cells. Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). The PVM plays an essential role for the interaction of parasites with their host cells, however only a limited number of proteins of this membrane have been identified so far. This is partially because a systematic proteomic analysis of the PVM’s protein content has been difficult in the past, due to difficulties to separate the PVM from other membranes such as the parasite plasma membrane. In this study, we adapted the BioID technique to in vitro cultivated Plasmodium berghei blood stage parasites and utilized the promiscuous biotin ligase BirA* fused to the PVM-resident exported protein 1 to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography based mass spectrometry (LC-MS/MS) and label-free quantitation leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000). In conclusion, our study expands the number of known PVM proteins and experimentally validates BioID as a powerful method to screen for novel constituents of specific cellular compartments in P. berghei.
2018-01-03 | PXD007857 | Pride
Project description:Recent advances in high throughput sequencing methodologies allow the opportunity to probe in depth the transcriptomes of organisms including N. caninum and Toxoplasma gondii. In this project, we are using Illumina sequencing technology to analyze the transcriptome (RNA-Seq) of experimentally accessible stages (e.g. tachyzoites at different times points) of T. gondii VEG strain. The aim is to make comparative transcriptional landscape maps of Neospora and Toxoplasma at different time points at different life cycle stages and compare levels of expression of orthologous genes in these two organisms.
2011-02-08 | E-MTAB-550 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE11437: Expression QTL mapping of Toxoplasma gondii genes, Bradyzoite array GSE11514: Expression QTL mapping of Toxoplasma gondii genes, Tachyzoite array Keywords: SuperSeries Refer to individual Series
2008-05-22 | E-GEOD-11515 | biostudies-arrayexpress