Expression data for various strains of Toxoplasma gondii chosen from canonical types (i.e., I,II, and III)
ABSTRACT: This study establishes a baseline pattern for RNA expression among canonical strains of Toxoplasma gondii grown in tissue culture without perturbation. Parasites cultured within human foreskin fibroblast (HFF) cells grown with D10 media were scraped and harvested ~8-12 hours prior to host cell lysis. RNA was isolated and applied to a T. gondii Affymetrix array.
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.
Project description:The in vitro effect of infection with different strains of Toxoplasma gondii was tested 24 hours after infection of Human Foreskin Fibroblasts (HFF) The strains tested include RH, VEG, and transgenic strains of RH overexpressing ROP38 or ROP21 Total RNA of Toxoplasma gondii infected HFF cell was compared to uninfected cells
Project description:Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome. Prugniaud, RH, and VEG strain parasites were cultured in human foreskin fibroblast (HFF) cells as previously described (Roos, Donald et al. 1994). Prior to host cell rupture, cells were scraped from the flask and spun at 300g for 9 min. The resultant pellet was lysed with Buffer RLT from the Qiagen RNeasy Mini Kit (Valencia, CA) and RNA was extracted according to the manufacturer’s instructions. Labeled cRNA was created using the One-Cycle Labeling protocol in the Affymetrix GeneChip® IVT Labeling Kit (Santa Clara, CA). Hybridization, washing, and scanning of arrays was performed using standard Affymetrix instrumentation and protocols for 11 micron, 169 format arrays. Biological triplicates were generated for each strain and expression values computed using the RMA implementation (default parameters) in the affy package from Bioconductor (Gentleman, Carey et al. 2004).
Project description:Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in recovering from alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. The TgGCN5-A knockout is incapable of up-regulating key marker genes expressed during development of the latent cyst form. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. We also describe a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that TgGCN5-A knockout parasites fail to up-regulate 68% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure, including developmentally regulated genes. Wild-type and GCN5-A- Knockout organisms were subjected to control and stress treatments
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.
Project description:The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages, types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between types I vs. III and II vs. III. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on a new genetic cross and linkage mapping. Genome-wide association revealed a single quantitative trait locus controls the > 4 log difference in lethality between these strains. Neither ROP16 nor ROP18, previously implicated in virulence differences in T. gondii, were found to contribute to differences between types I and II. Instead, the major virulence locus contained a cluster of pseudokinases denoted as rhoptry protein 5 (ROP5); this locus contains a tandem cluster of polymorphic alleles that differed in expression levels between strains. ROP5 alleles contained only part of the catalytic triad of canonical S/T kinases, and consistent with this they lack demonstrable kinase activity in vitro. Genetic disruption of the rop5 locus in the type I lineage lead to a > 5 log increase in the lethal dose, and surviving mice developed lasting immunity and were protected from an otherwise lethal challenge. These findings reveal that amplification of a polymorphic cluster of pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model. We used a new genotyping protocol based on hybridization of parental and recombinant progeny genomic DNA (gDNA) to Affymetrix gene arrays constructed for T. gondii (http://ancillary.toxodb.org/docs/Array-Tutorial.html). We identified 1,603 single feature polymorphisms (SFP) based on probes that successfully discriminated perfect match (type II) from mismatch (type I) hybridization across all clones and provided reliable SFP markers for this cross.
Project description:Toxoplasma gondii is an obligate intracellular protozoan parasite whose rapid lytic replication cycles define its pathogenicity. We identified a temperature sensitive growth mutant, FV-P6, which irreversibly arrests before the middle of the G1 stage of the tachyzoite cell cycle. This arrest is caused by a point mutation in a gene conserved across eukaryotes, Cactin, whose product localizes to the nucleus. To elucidate the role of TgCactin we performed genome-wide expression profiling of FV-P6 mutant parasites at 35C and 40C, as well as FV-P6 complemented with a wild-type Cactin encoding cosmid (TOXO93 or comp) and as a pseudo-diploid wild-type line encoding both the wild-type and mutant Cactin locus (S4.2), also at both temperatures. Besides the expected G1 expression profile, many genes associated with the extracellular state as well as with the bradyzoite cyst stage were identified. Consistent with these profiles were the expression of AP2 transcription factors typically associated with extracellular and bradyzoite stage parasites. This suggests a role for TgCactin in control of gene expression. Since TgCactin does not contain any functionally defined domains we reasoned TgCactin exerts its function through interactions with other proteins. In support of this model we demonstrated that TgCactin is present in a protein complex and can oligomerize. Taken together, these results suggest that TgCactin acts as a pivotal protein potentially regulating gene expression at several transition points in parasite development. The role of Cactin in G1 progression of Toxoplasma gondii tachyzoites was assessed by expression profiling a temperature senstive mutant of Cactin at both the permissive (35C) as well as the restrictive (40C) temperature. Controls under the same temperature conditions inlcude the mutant line complemented with a wild-type Cactin allele encoded on a cosmid and a wild-type parasite line expressing an additional, mutant allele of Cactin (pseudo-diploid line).
Project description:PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. We generated T. gondii mutants lacking PRMT1 (∆prmt1) by deletion of the PRMT1 gene. ∆prmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the complemented strain ∆prmt::PRMT1mRFP. PRMT1 localizes primarily in the cytoplasm with enrichment at the centrosome, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, ∆prmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole genome expression profiling demonstrated differences in expression of cell cycle regulated genes in ∆prmt1 relative to the complemented ∆prmt1::PRMT1mRFP and parental wild-type strains, but these changes did not correlate with a specific block in cell cycle. Although PRMT1’s primary biological function was previously proposed to be methylation of histones, our genetic studies suggest that the most critical function of PRMT1 is within the centrosome as a regulator of daughter cell counting to assure the proper replication of the parasite. RNA samples were isolated in triplicates from RH-hxgprt parent strain (W), PRMT1 knockout (K) strain and PRMT1 knockout strain complemented with RFP-tagged PRMT1 protein (C). Parasites were grown for 32h at 37C. Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies).
Project description:Toxoplasma gondii temperature sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. We describe transcriptome of the temperature sensitive mutant 12-109C6. Transcriptome studies demonstrated that gene expression is largely downregulated in the mutant at the restrictive temperature 40oC and is accompanied by a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. RNA samples were isolated in duplicate from mutant 12-109C6 parasites grown for 24h at permissive (34oC) and 6h and 24h at non-permissive temperatures (40oC). Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies, Santa Clara CA).
Project description:The parents and progeny of the I X III genetic cross were genotyped using the ToxoGeneChip in order to generate a more detailed genetic map. We also used the genomic hybridization data to look for copy number variations (CNV) and segmental duplications. The ToxoGeneChip microarray (http://ancillary.toxodb.org/docs/Array-Tutorial.html) was used to hybridize genomic DNA for the parents and progeny of the I X III cross. The parents of the cross are CTGara and GT1-Fudr. The rest comprise the 34 informative progeny.