In vivo CRISPR screens for Toxoplasma using ROP/GRA sublibrary in WT and Ifngr1-deficient mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Toxoplasma gondii
PROVIDER: GSE229545 | GEO | 2023/06/12
REPOSITORIES: GEO
Similar Datasets
Project description:We performed in vivo CRISPR screen of Toxoplasma targeting metabolism-related genes to identify in vivo fitness genes in WT and Ifngr1-deficient mice.
2023-06-12 | GSE229548 | GEO
Project description:We performed in vivo CRISPR screen of Toxoplasma targeting endomembrane and nucleus-related genes to identify in vivo fitness genes in WT and Ifngr1-deficient mice.
2023-06-12 | GSE229547 | GEO
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.
2018-01-31 | GSE109830 | GEO
Project description:Toxoplasma gondii is an intracellular parasite that infects all nucleated cells; to date, this organism is considered as one of the most successful parasites in the world. During infection, internalization, and proliferation the parasite excretes/secretes a high number of proteins, several of them with identified roles, including MIC proteins, ROP proteins and GRA proteins involved in adhesion, invasion and intracellular proliferation respectively. The characterization of the secretome of Toxoplasma gondii opens the possibility to find new molecules for the design of future anti-parasitic strategies. The complete secretome and proteome of the parasite isolated from the murine model as well as their quantification by MS analysis are now available in the present report.
2019-08-29 | PXD013767 | Pride
Project description:Differential impacts on host transcription by ROP and GRA effectors from the intracellular parasite Toxoplasma gondii
| PRJNA608271 | ENA
Project description:Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense granule proteins (GRA) are important effectors that mediate host-parasite interactions and facilitate parasitism. Due to the wide host range, T. gondii needs diverse GRA proteins to accommodate parasite survival in different hosts. In this study, we used two proximity based protein labeling techniques to identify novel GRA proteins. Using GRA1 as bait, two transgenic strains expressing GRA1-BirA* or GRA1-APEX were used to biotinylate GRA candidate proteins. Using these methods, 90 proteins were identified by APEX based labeling and 56 by the BirA* approach. Twenty-three were identified by both techniques, among which 13 are known GRA proteins. In combination 98 novel GRA candidates were identified by these two approaches and 25 are known GRAs. The majority of them are only present in coccidian parasites and dispensable for parasite growth in vitro, they likely mediate interactions with hosts and play roles during animal infections.
2018-11-02 | PXD009604 | Pride
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:The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context- dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell. Additional data can be found at tko.ccbr.utoronto.ca RNAseq of five human cell lines with Cas9 knock-ins.
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Project description:Investigate the effect of depleting Ifngr1 in microglia on their response to acute neurodegeneration
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Project description:The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system. polyA RNA profiling of Neural Stem/Progenitor cells (NPCs) cultured in basal/Th1/Th2 conditions, of Exosomes derived from NPCs cultured in basal/Th1/Th2 conditions and of EVs derived from NPCs cultured in Basal/Th1/Th2 conditions. Total RNA was purified using Trizol. Purity and integrity were confirmed by BioAnalyser (Agilent). Paired End library construction and poly-A selection were performed by EASIH (The Eastern Sequence and Informatics Hub, University of Cambridge, Cambridge) according to the Illumina standard protocol. Sequencing was performed by EASIH using Illumina GAII.
2014-09-22 | E-GEOD-33527 | biostudies-arrayexpress