Analysis of the role for TgAP2IX-4 in regulating gene expression in Toxoplasma development
Ontology highlight
ABSTRACT:
ORGANISM(S): Toxoplasma gondii
PROVIDER: GSE94045 | GEO | 2017/01/26
SECONDARY ACCESSION(S): PRJNA368637
REPOSITORIES: GEO
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Project description:Differential gene expression analysis of TgAP2IV-4 knockout parasites in type I RH diCRE (RHCre) and type II Prudku80 (PruQ) genetic background The gene regulatory role for TgAP2IV-4 was evaluated, in vitro, as tachyzoites (pH 7.0) in 8 strains: RHCre-AP2IV-4 floxed; RHCre-delta_ap2IV-4 clones 27 and 30, PSMB794 cosmid complemented RHCre-dAP2IV-4 populations; PruQ-AP2IV-4 floxed; PruQ-delta_ap2IV-4 clones 10 and 34.
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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
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Project description:Thiouracil Pulse-chase, Tachyzoites and Bradyzoites mRNA stability data
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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:Using a differentiation reporter strain, unstressed parasites (tachyzoites) or stressed, reporter positive parasites (bradyzoites) were isolated by flow cytometry and analyzed for differential gene expression.
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Project description:The purpose of this data set is to determine if bradyzoites impact the host transcripton in a similar manner as tachyzoites. We found that 3853 differentially expressed genes (DEGs) are shared between tachyzoite infected cells and bradyzoite infected cells (41%; 2161 up-reg., 1692 down-reg.) when compared to uninfected host samples, leaving 3356 tachyzoite specific DEGs (1488 up-reg., 1868 down-reg.), and 1951 DEGs specific to bradyzoite infection (1075 up-reg., 876 down-reg). We conclude therefore that bradyzoites manipulate the host cell and that this is unique when compared to tachyzoites.
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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: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.
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Project description:The Toxoplasma gondii G1 RESTRICTION checkpoint operates the switch between parasite growth and differentiation. The Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2 and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. The TgCycP1 expression interferes with bradyzoite differentiation. The TgCycP2 regulates G1 in the developmentally competent ME49 but not in the developmentally incompetent RH tachyzoites. Examination of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models confirmed TgCycP2 role in bradyzoite replication and revealed that stress induced TgCyc5 is critical for efficient tissue cyst maturation.
2022-04-23 | GSE200962 | GEO
Project description:Although apicomplexans lack conventional R-point regulators, presence of the quiescent G1 forms of 43 parasites, including mature T. gondii bradyzoites suggests that a functional RESTRICTION checkpoint 44 exists in apicomplexan parasites. We showed that Cdk-related G1 kinase TgCrk2 forms alternative 45 complexes with atypical cyclins (TgCycP1, TgCycP2 and TgCyc5) in the rapidly dividing developmentally 46 incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. 47 Examination of individual cyclins verified the correlation of cyclin expression with growth dependence and 48 development capacity of type I RH and type II ME49 strains. We demonstrated that rapidly dividing RH 49 tachyzoites are dependent on TgCycP1 and expression of TgCycP1 interferes with bradyzoite 50 differentiation. By using an auxin-induced degradation (AID) model, we established that TgCycP2 51 regulates G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally 52 incompetent RH tachyzoites. We also tested the functions of TgCycP2 and TgCyc5 in alkaline induced in 53 vitro model and in ex vivo model of spontaneous bradyzoite differentiation (rat embryonic brain cells). 54 Based on functional and global gene expression analyses, we determined that TgCycP2 also regulates 55 bradyzoite replication, while stress- or host-induced TgCyc5 was critical for efficient bradyzoite conversion 56 and tissue cyst maturation.
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