RNA sequencing of host transcriptome of human foreskin fibroblast cells infected with either bradyzoites and tachyzoite of Toxoplasma gondii
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ABSTRACT:
ORGANISM(S): Toxoplasma gondii Homo sapiens
PROVIDER: GSE125121 | GEO | 2020/01/15
REPOSITORIES: GEO
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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.
2022-07-23 | GSE208647 | GEO
Project description:Parasites grown as either tachyzoites or bradyzoites for 40 hours were pulsed with 2,4-dithiouracil (DTU) for 1 hour. Following the pulse, mRNA was extracted and either used directly in microarray experiments (Abundance Arrays) or biotinylated and purified to select DTU labeled RNAs that were then used in microarray experiments (Synthesis Arrays). Two independent tachyzoite and bradyzoite preparations were made and duplicate microarrays were performed for each sample.
2005-07-07 | E-SMDB-2373 | biostudies-arrayexpress
Project description:Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identify 241 genes differentially expressed between CTG tachyzoites and in vitro bradyzoites, including 5 putative AP2 transcription factors. We also observe close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysis (GSEA), there are a number of KEGG pathways associated with the in vitro bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identify 1,526 genes that are differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identify ribosomal proteins as being expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage. Type I M-bM-^@M-^S RH, Type II M-bM-^@M-^S PLK, and Type III CTG parasites were each grown in either alkaline, bradyzoite conditions or pH neutral, tachyzoites conditions. 6 conditions, 3 replicates each, 18 total samples
2014-09-01 | E-GEOD-60305 | biostudies-arrayexpress
Project description:Using high through-put RNA sequencing, we assayed the transcriptomes of three different strains of Toxoplasma gondii representing three common genotypes under both in vitro tachyzoite and in vitro bradyzoite-inducing alkaline stress culture conditions. Strikingly, the differences in transcriptional profiles between the strains, RH, PLK, and CTG, is much greater than differences between tachyzoites and alkaline stressed in vitro bradyzoites. With an FDR of 10%, we identify 241 genes differentially expressed between CTG tachyzoites and in vitro bradyzoites, including 5 putative AP2 transcription factors. We also observe close association between cell cycle regulated genes and differentiation. By Gene Set Enrichment Analysis (GSEA), there are a number of KEGG pathways associated with the in vitro bradyzoite transcriptomes of PLK and CTG, including pyrimidine metabolism and DNA replication. These functions are likely associated with cell-cycle arrest. When comparing mRNA levels between strains, we identify 1,526 genes that are differentially expressed regardless of culture-condition as well as 846 differentially expressed only in bradyzoites and 542 differentially expressed only in tachyzoites between at least two strains. Using GSEA, we identify ribosomal proteins as being expressed at significantly higher levels in the CTG strain than in either the RH or PLK strains. This association holds true regardless of life cycle stage.
2014-09-01 | GSE60305 | GEO
Project description:Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions.
2005-07-21 | E-SMDB-1406 | biostudies-arrayexpress
Project description:Pulse chase measurements using thiouracil (DTU) labeling via UPRT and chasing with uracil Data from tachyzoites is labeled "DTU Pulse Chase". Two independent pulse chase experiments were performed in tachyzoites, pulse chase 1 and 2. Duplicate arrays at each timepoint were performed for pulse chase 2 (2 a and b). Data from bradyzoites are labeled "DTU Bradyzoite Pulse Chase". Two independent pulse chase experiments were performed in bradyzoites and a single set of arrays were performed for each experiment. Just one chase timepoint was used in the bradyzoite experiments, the 2 hour chase.
2005-07-07 | E-SMDB-2949 | biostudies-arrayexpress
Project description:Ingestion of Toxoplasma gondii results in life-long infection due to its ability to convert from the rapidly disseminating tachyzoite stage to the chronic, encysted bradyzoite stage. The control of mRNA translation has been suggested to play a key role in the signaling required to trigger bradyzoite formation. In eukaryotes, translational control primarily operates at two key points during the assembly of translation initiation factors. The phosphorylation of eIF2 affects mRNA start codon recognition and promotes differentiation in a variety of parasites. Modulating eIF4F function is the second pan-eukaryote regulatory point but remains unexplored in Toxoplasma. Here, we uncover the role that eIF4F-centric regulation plays in regulating bradyzoite formation by targeting the cap-binding subunit, eIF4E1. We discover that eIF4E1 coordinates two eIF4F complexes and binds the 5’-end of all mRNAs transcribed in tachyzoites, many of which show evidence of stemming from heterogenous transcriptional start sites. Together, this indicates that eIF4E1 is the predominant cap-binding protein in Toxoplasma tachyzoites. We also demonstrate that eIF4E1 knockdown or its chemical inhibition triggers the efficient formation of bradyzoites in the absence of other stresses and that stress-induced bradyzoites reduce their eIF4E1 expression. This study unearths a role for eIF4F-centric translational control in controlling Toxoplasma differentiation, suggesting that control of cap-dependent translation regulates the process of bradyzoite formation.
2023-11-02 | GSE243206 | GEO
Project description:Ingestion of Toxoplasma gondii results in life-long infection due to its ability to convert from the rapidly disseminating tachyzoite stage to the chronic, encysted bradyzoite stage. The control of mRNA translation has been suggested to play a key role in the signaling required to trigger bradyzoite formation. In eukaryotes, translational control primarily operates at two key points during the assembly of translation initiation factors. The phosphorylation of eIF2 affects mRNA start codon recognition and promotes differentiation in a variety of parasites. Modulating eIF4F function is the second pan-eukaryote regulatory point but remains unexplored in Toxoplasma. Here, we uncover the role that eIF4F-centric regulation plays in regulating bradyzoite formation by targeting the cap-binding subunit, eIF4E1. We discover that eIF4E1 coordinates two eIF4F complexes and binds the 5’-end of all mRNAs transcribed in tachyzoites, many of which show evidence of stemming from heterogenous transcriptional start sites. Together, this indicates that eIF4E1 is the predominant cap-binding protein in Toxoplasma tachyzoites. We also demonstrate that eIF4E1 knockdown or its chemical inhibition triggers the efficient formation of bradyzoites in the absence of other stresses and that stress-induced bradyzoites reduce their eIF4E1 expression. This study unearths a role for eIF4F-centric translational control in controlling Toxoplasma differentiation, suggesting that control of cap-dependent translation regulates the process of bradyzoite formation.
2023-11-02 | GSE243203 | GEO
Project description:Two forms of the protozoan parasite Toxoplasma gondii are associated with intermediate hosts such as humans: rapidly growing tachyzoites are responsible for acute illness, whereas slowly dividing encysted bradyzoites can remain latent within the tissues for the life of the host. In order to identify genetic factors associated with parasite differentiation, we have used a strong bradyzoite-specific promoter (identified by promoter trapping) to drive the expression of T. gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) in stable transgenic parasites, providing a stage-specific positive/negative selectable marker. Insertional mutagenesis has been carried out on this parental line, followed by bradyzoite induction in vitro and selection in 6-thioxanthine to identify misregulation mutants. Two different mutants fail to induce the HXGPRT gene efficiently during bradyzoite differentiation. These mutants are also defective in other aspects of differentiation: they replicate well under bradyzoite growth conditions, lysing the host cell monolayer as effectively as tachyzoites. Expression of the major bradyzoite antigen BAG1 is reduced, and staining with Dolichos biflorus lectin shows reduced cyst wall formation. Microarray hybridizations show that these mutants behave more like tachyzoites at a global level, even under bradyzoite differentiation conditions. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. User Defined
2005-08-13 | E-GEOD-3119 | biostudies-arrayexpress
Project description:Parasites grown as either tachyzoites or bradyzoites for 40 hours were pulsed with 2,4-dithiouracil (DTU) for 1 hour. Following the pulse, mRNA was extracted and either used directly in microarray experiments (Abundance Arrays) or biotinylated and purified to select DTU labeled RNAs that were then used in microarray experiments (Synthesis Arrays). Two independent tachyzoite and bradyzoite preparations were made and duplicate microarrays were performed for each sample. A development or differentiation experiment design type assays events associated with development or differentiation or moving through a life cycle. Development applies to organism(s) acquiring a mature state, and differentiation applies to cells acquiring specialized functions. User Defined
2005-07-16 | E-GEOD-2946 | biostudies-arrayexpress