In vitro analysis of pH responsiveness of T. gondii and H. hammondi sporozoites, and of T. gondii tachyzoites lacking either the BFD1 or the ROCY1 gene.
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ABSTRACT:
ORGANISM(S): Toxoplasma gondii Hammondia hammondi
PROVIDER: GSE218921 | GEO | 2023/08/14
REPOSITORIES: GEO
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Project description:Toxoplasma gondii pathogenesis includes the invasion of host cells by extracellular parasites (tachyzoites), replication of intracellular tachyzoites, and differentiation to a latent bradyzoite stage. Whole genome expression profiling was carried out using the newly developed Affymetrix ToxoGeneChip (GeneChip Tgondiia520372) in order to analyze the ~8,000 predicted genes in the T. gondii genome of mutants and wild-type, allowing for full-scale expression profiling during bradyzoite differentiation in vitro. RNA from mutant and wild-type parasites was extracted and hybridized to the ToxoGeneChip. We harvested extracellular tachyzoites from freshly lysed fibroblasts (ET, 0h), intracellular tachyzoites (IT, 24h post-invasion) and parasites subjected to bradyzoite growth conditions for 72h (B72, 72h of induction). Extracellular parasites from freshly lysed fibroblasts were harvested for the seven mutant parasite lines (12K, 13P, B7, 11P, 11K, 7K and P11) and mutant parasites subjected to bradyzoite differentiation conditions for 72h. For a few samples we also harvested parasites 11h post egress of host cells. A time course was carried out with wild-type: parasites subjected to bradyzoite growth conditions for 24h, 36h and 48h.
2010-07-27 | E-GEOD-23174 | biostudies-arrayexpress
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: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: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. Keywords: Logical Set
2005-08-13 | GSE3119 | GEO
Project description:Two samples, 0hr and 72hr, were used to generate tachyzoite and bradyzoite transcriptional data from tissue-cultured Toxoplasma gondii strain Prugniaud, respectively. Samples are single replicates, and a subset of a larger timeseries. Non-control sample was exposed to alkaline conditions, media pH 8.2, for 72hr.
2010-06-15 | E-GEOD-22258 | biostudies-arrayexpress
Project description:tRNAs are critical linker molecules that couple mRNA to protein translation and their availability is a limiting factor for translation. tRNA transcription and the composition of the tRNA pool are both dynamically regulated in response to environmental cues. Recent work suggests that they also play an important role in regulating cell growth and differentiation. Toxoplasma gondii is a highly adaptable, early eukaryotic protozoan that is able to replicate in virtually any nucleated cell and many different host organisms, thus requiring fine-tuning of protein expression. A key feature of parasite pathogenesis is the ability to differentiate between fast growing tachyzoite and slow growing bradyzoite cyst forms, in response to stress e.g. nutrient starvation. Differentiation is accompanied by changes to the mRNA transcriptome and proteome but little is known about translational regulation. In this study, we used comparative genomics to catalogue the repertoire of tRNA genes in T. gondii and their genomic distribution. Interestingly, many tRNAs occur in regulatory regions and intronic sequences of protein-coding genes, which correlates with increased gene expression. To characterize the tRNA pool of T. gondii during stress and differentiation, we surveyed levels of precursor tRNA molecules in the tRNA population by small RNA-seq in three divergent parasite strains with varying bradyzoite differentiation competencies, cultured in normal or bradyzoite differentiation-inducing (pH shock) conditions. Almost all tRNA genes are constitutively transcribed in T. gondii tachyzoites and transcription of individual tRNA genes differs significantly between strains. The abundance of individual tRNAs in the tRNA pool reflects proteomic codon usage, suggesting that tRNA levels are tightly controlled according to the demands of translation. Together, this work suggests that modulation of the tRNA pool is a mechanism by which the protein landscape in T. gondii is regulated in different strains and developmental stages.
2017-09-08 | GSE86616 | GEO
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:Background: Considerable work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, of Toxoplasma gondii in large part due to the accessible culturing methods for these stages. However, culturing methods for stages beyond the bradyzoite, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. We begin to unravel the molecular aspects of the merozoite stage focusing on gene expression. Results: To initiate this, we harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by combining it with a previously published study that generated array data for the oocyst, tachyzoite, and bradyzoite stages (Fritz HM et al. PLoS One, 2012). Principal component analysis highlights the unique profile of the merozoite samples, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen p30 (SAG1) and many dense granule proteins do not label merozoites, and our microarray data confirms that these genes are not expressed at this stage. Also, the expression for many rhoptry and microneme proteins is drastically reduced while the expression for many surface antigens is increased at the merozoite stage. Gene Ontology and KEGG analysis reveals that genes involved in transcription/translation and many metabolic pathways are upregulated at the merozoite stage, highlighting unique growth requirements of this stage. We also show that an upstream promoter region of a merozoite specific gene is sufficient to control stage specific expression at the merozoite stage. Conclusion: The merozoite represents the first developmental stage within the gut of the definitive host. Determining the correct conditions that coax the parasite into the merozoite stage in vitro may allow the parasite to complete sexual development. The data presented here describe the global gene expression profile of merozoite stage and the creation of transgenic parasite strains that will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate coccidian development. The ToxoGeneChip microarray was used to measure both tachyzoite and merozoite mRNA expression in the type II TgNmBr1 strain.
2014-05-02 | E-GEOD-51780 | biostudies-arrayexpress
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.
2019-07-11 | GSE134099 | GEO