The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections.
Ontology highlight
ABSTRACT:
ORGANISM(S): Toxoplasma Gondii
TISSUE(S): Cultured Cells
DISEASE(S): Parasitic Infection
SUBMITTER: 
Martin Blume
PROVIDER: ST002051 | MetabolomicsWorkbench | Tue Jan 04 00:00:00 GMT 2022
REPOSITORIES: MetabolomicsWorkbench
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Project description:The apicomplexan parasite Toxoplasma gondii forms bradyzoite-containing tissue cysts that cause chronic and drug-tolerant infections. Here, we developed a human myotube-based in vitro culture model of functionally mature tissue cysts. In contrast to fast replicating tachyzoite forms of T. gondii these tissue cysts tolerate exposure to the aconitase inhibitor sodium fluoroacetate. These data characterize bradyzoites and tachyzoites treated with NaFAc.
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Project description:Toxoplasma gondii is a zoonotic pathogen for which felids serve as definitive hosts. In cats, the parasite undergoes several rounds of asexual replication before entering the sexual cycle which gives rise to oocysts that are shed into the environment. These then sporulate and become infective to humans and live stock. To understand the genes involved in the parasite development in the felid host and identify potential intervention targets, we designed a transcriptomic approach to compare the cat intestinal stages with the well characterised tachyzoites that mediate acute infection and tissue cysts that are responsible for chronic infection. Cats were infected with T. gondii tissue cysts from mouse brain and sampled the intestinal stages at day 3, 5 and 7 post infection. As an input sample, we also collected tissue cysts from mouse brain as well as in vitro cultivated tachyzoites. Total RNA was extracted, enriched for mRNA and used for cDNA synthesis. RNA-Seq was then performed to describe the transcriptomic repertoire of each time point/life cycle stage.
2018-03-30 | GSE108740 | GEO
Project description:The protozoan pathogen Toxoplasma gondii has the unique ability to develop a chronic infection in the brain of its host. The T. gondii develops cysts within the neurons that are resilient against the host immune response and current therapeutics. The bradyzoite parasites within the cyst have a sugar-protein-rich wall and a slow-replication cycle, allowing them to remain hidden from the host. This intracellular encysted lifestyle of T. gondii has made them recalcitrant to molecular analysis in vivo. Here, we have enriched for T. gondii brain cysts 21 to 150 days post-infection (DPI). RNA and protein from bradyzoites was isolated from each timepoint. Deep sequencing of transcripts expressed during these three timepoints showed many of the identified proteins are transcriptionally expressed at a high level. Approximately one-third are more enriched during bradyzoite conditions compared to tachyzoites, and approximately half are expressed at similar levels during each phase. We have expanded the transcriptional profile of in vivo bradyzoites to 120 DPI. The RNA sequencing depth of in vivo bradyzoite T. gondii was over 250-fold greater than was have been previously, which allowed us to identify low level transcripts and novel bradyzoite-specific isoforms.
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Project description:Toxoplasma gondii is a eukaryotic parasite that form latent cyst in the brain of immunocompetent individuals. The latent parasites infection of the immune privileged central nervous system is linked to most complications. With no drug currently available to eliminate the latent cysts in the brain of infected hosts, the consequences of neurons long-term infection are unknown.. It has long been known that T. gondii specifically differentiate into a latent form (bradyzoite) in neurons, but how the infected neuron is responding to the infection remain to be elucidated. We have established a new in vitro model resulting in the production of fully mature bradyzoites cysts in brain cells. Using dual, host and parasite, RNA-seq we characterized the dynamics of differentiation of the parasite, revealing the involvement of key pathways in this process. Moreover, we identified how the infected brain cells responded to the parasite infection revealing the drastic changes that take place. We showed that neuronal specific pathways are strongly affected, with synapse signaling being particularly affected, especially glutamatergic synapse. The establishment of this new in vitro model allows to investigate both the dynamics of the parasite differentiation and the specific response of neurons to the long term infection by this parasite.
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Project description:Cyst formation is a key feature of the T. gondii life cycle but the genetic networks that drive this process are not yet fully characterized. To identify new components of this network, we compared T. gondii to its nearest extant relative Hammondia hammondi given the critical differences between these species in the timing and efficiency of cyst formation. Using transcriptional data from critical developmental and pH exposure time points from both species, we identified the gene TGVEG_311100, which we named Regulator of Cystogenesis 1 (ROCY1), as being both necessary and sufficient for cyst formation in T. gondii. Compared to WT parasites, TGVEG?ROCY1 parasites formed significantly fewer tissue cysts in response to alkaline pH stress in vitro and cysts were nearly undetectable in mouse brains for up to 9 weeks post-infection. Overexpression of tagged ROCY1 in WT parasites was sufficient to induce cyst formation in vitro in both WT and ROCY1-deficient parasites, demonstrating that ROCY1 is both necessary and sufficient for cyst formation. Moreover this induction of cyst formation required at least 1 of 3 predicted CCCH Zinc finger domains. Mice chronically infected with ?ROCY1 parasites had detectable tachyzoites in the brain for up to 37 days post-infection (while mice infected with WT parasites did not), and CNS transcriptional analyses at day 30 post-infection throughout the chronic phase of infection revealed inflammatory signatures consistent with acute infection in ?ROCY1 parasites compared to WT. Despite our inability to detect brain cysts in infected mice, both WT and ?ROCY1 knockout parasites reactivated after dexamethasone treatment with similar timing and magnitude for up to 5 months post infection, challenging the paradigm that long term parasite persistence in the CNS requires cyst formation. These data identify a new regulator of cyst formation in T. gondii that is both necessary and sufficient for cyst formation, and whose function relies on its conserved nucleic acid binding motif.
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Project description:Toxoplasma has been a useful parasite model for decades because it is relatively easy to genetically modify and culture, however, attempts to generate and study the recrudescence of tissue cysts have come up short with lab-adapted strains generating low numbers of tissue cysts in vivo. Here we have established a new model of Toxoplasma recrudescence using bradyzoites from an unadapted Type II ME49 strain (ME49EW) isolated from murine brain tissue. Ex vivo bradyzoite infection of fibroblasts and astrocytes produced sequential tachyzoite growth stages; a fast-growing stage was followed by formation of a slower-growing stage. In astrocytes, but not in fibroblasts, bradyzoites also initiated a second recrudescent pathway involving bradyzoite to bradyzoite replication. Intraperitoneal infections of mice with either bradyzoites or the fast-growing tachyzoite stage efficiently disseminated to brain tissue leading to high numbers of tissue cysts, while infections with the slow-growing tachyzoite stage were largely retained in the peritoneum. Poor infection and cyst formation of slow-growing tachyzoites was reversible by serial tissue cyst passage, while the poor tissue cyst formation of lab-adapted tachyzoites was not reversible by these approaches. To distinguish strain developmental competency, we identified Toxoplasma genes highly expressed in ME49EW in vivo tissue cysts and developed a qPCR approach that differentiates immature from mature bradyzoites. In summary, the results presented describe a new ex vivo bradyzoite recrudescence model that fully captures the growth and developmental processes during toxoplasmosis reactivation in vivo opening the door to the further study of these important features of the Toxoplasma intermediate life cycle.
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Project description:Purpose: Porcine alveolar macrophage was infected by T. gondii including Rh strain and Me49 strain. We want to explore the change of miRNAs after infected with T. gondii in porcine alveolar macrophages. Results: Our study generated six mi RNA expression profiles from macrophages which infect with Rh strain and Me49 and control group in different time. Compare with T. gondii-infected and uninfected with T. gondii, 81 differentially expressed mi RNAs were identified, including 36 novel mi RNAs and 45 mature mi RNAs.
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Project description:Toxoplasma gondii is an obligatory intracellular parasite that causes persistent infections in birds and mammals including ~30% of the world’s human population. Differentiation from proliferative and metabolically active tachyzoites to largely dormant bradyzoites initiates the chronic phase of infection and occurs predominantly in brain and muscle tissues. Here we used murine skeletal muscle cells (SkMCs) to decipher host cellular factors that favor T. gondii bradyzoite formation in terminally differentiated and syncytial myotubes, but not in proliferating myoblast precursors. Genome-wide transcriptome analyses of T. gondii-infected SkMCs and non-infected controls identified ~6,500 genes which were differentially expressed (DEGs) in myotubes compared to myoblasts, largely irrespective of infection. On the other hand, genes related to central carbohydrate metabolism, to redox homeostasis, and to the Nrf2-dependent stress response pathway were enriched in both infected myoblast precursors and myotubes. Stable isotope-resolved metabolite profiling indicated increased fluxes into the oxidative branch of the pentose phosphate pathway (OxPPP) in infected myoblasts and into the TCA cycle in infected myotubes. High OxPPP activity in infected myoblasts was associated with increased NADPH/NADP+ ratio while myotubes exhibited higher ROS levels and lower expression of anti-oxidants and detoxification enzymes. Pharmacological reduction of ROS levels in SkMCs inhibited bradyzoite differentiation, while increased ROS induced bradyzoite formation. Thus, we identified a novel host cell-dependent mechanism that triggers stage conversion of T. gondii into persistent tissue cysts in its natural host cell type.
2021-11-04 | GSE133952 | GEO
Project description:MOB1 is a conserved protein that regulates cellular proliferation versus apoptosis, centrosome duplication and cellular differentiation in multicellular eukaryotes and also cytokinesis and division axis orientation in unicellular and multicellular eukaryotes. Toxoplasma gondii, an obligate intracellular parasite of veterinary and medical importance, presents one MOB1 protein. T. gondii interconverts between several cellular stages during its life cycle, namely between fast replicating tachyzoite and slow replicating bradyzoite stages during its asexual cycle, a key ability for its success as a parasite. Bradyzoites produce tissue cysts, establishing a chronic infection that enables recrudescence. Conversion is dependent on cell cycle regulation and involves cell differentiation and regulation of replication. This led us to select MOB1 as a strong candidate to be involved in the Toxoplasma replication process. To elucidate how MOB1 acts in T. gondii, we employed a proximity biotinylation method and identified the MOB1 interactome. Toxoplasma gondii RH tachyzoites were transfected with BirA containing plasmid vectors for random integration and two strains were isolated. The control strain expresses a FLAG-BirA recombinant protein while the test strain expresses a FLAG-BirA-MOB1 recombinant protein. Biotinylated proteins were purified using streptavidin-agarose beads. The purified proteins were trypsinized and analyzed by nanoLC-MS/MS.
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Project description:The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5’-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.
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