Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction

Abstract

Summary Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd−) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd− mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite‐inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild‐type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite‐induced genes whose transcripts were reduced in all mutants. A set of non‐developmentally regulated genes whose transcripts were less abundant in Tbd− mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite‐induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single‐celled eukaryotes including protozoa and fungi.

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

“…We note that these TgPUS1-deficient parasites are insertional mutants (TBD5, isolated by Singh et al 2002 and characterized by Anderson et al 2009), rather than TgPUS1 knockouts. Furthermore TgPUS1-deficient parasites incubated under bradyzoite conditions for 84 h are not able to differentiate; therefore, TgPUS1-deficient "bradyzoites" express subsets of both tachyzoite and bradyzoite markers (Anderson et al 2009 and data not shown).…”

“…To begin the identification of Ψ's in Toxoplasma, i.e., its predicted "pseudouridylome," we performed PSI-seq on tachyzoite and bradyzoite RNA isolated from wild-type parasites (specifically C32 parasites, a clone derived from a Type II Pru strain [Singh et al 2002]). Tachyzoites were grown in neutral pH and serum-rich conditions, while bradyzoite differentiation was induced using starvation (1% FBS) and high pH (8.0).…”

“…Parasite strains C32 (a clone derived from a type II Pru strain of Toxoplasma expressing GFP under the bradyzoitespecific LDH2 promoter) and TBD5 (a mutant made in the C32 strain that contains an insertion in the PUS1 gene [TgME49_ 202640]) were originally isolated as described in Singh et al (2002). Toxoplasma gondii tachyzoites were maintained by serial passage in confluent HFFs and kept at 37°C with 5% CO 2 .…”

“…This single-celled eukaryote can exist within its human host as fast-growing tachyzoites or as slower-growing encysted bradyzoites. In two independent screens for Toxoplasma mutants that are defective in differentiation from tachyzoites to bradyzoites, we found mutants with disruption of a predicted PUS (TGME49_202640) (Singh et al 2002;Anderson et al 2009). This protein was named TgPUS1, as it was the first PUS investigated in Toxoplasma, and not due to any specific homology between TgPUS1 and yeast PUS1.…”

Cyclic nucleotide kinases and tachyzoite–bradyzoite transition in Toxoplasma gondii

“…This method of measuring differentiation is laborious and has numerous steps in which experimental variation could mask significant but subtle results. Flow cytometry can also be used to assess bradyzoite differentiation in parasites expressing GFP driven by a bradyzoite-specific promoter such as LDH2 but this method does not quantitate growth in parallel (Singh et al, 2002). Currently, there is no parasite that allows enzymatic quantitation of both growth and bradyzoite gene expression.…”