A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation inToxoplasma gondii

Abstract

Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd ؊ ) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5 end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (⌬pus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only ϳ10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.Toxoplasma gondii is an apicomplexan parasite that is unique in its wide host range and global presence. In humans, the prevalence rate ranges from 15% to 75%, depending on geographic region (26). This high prevalence is due, in part, to the ability of the parasite to efficiently initiate an infection using either of two developmental stages, each with its own mode of transmission. Chronic infection is also a result of the ability of Toxoplasma to successfully evade clearance by the host immune system. All of these phenomena are dependent on the complex but well-described developmental biology of this coccidian parasite.Toxoplasma disseminates within a host primarily through interconversion between two asexual stages, the tachyzoite and bradyzoite forms. One way that a new host can be infected is through ingestion of raw or undercooked tissue from an infected animal. Bradyzoite-containing cysts in the tissue break open due to the pepsin and acidic environment of the digestive tract, after which the bradyzoites within are released and invade through the intestinal epithelium. These bradyzoites quickly convert into tachyzoites, the rapidly dividing, diseasecausing form of the parasite, which spread throughout the infected individual. During parasite expansion, adaptive immunity is triggered; this efficiently clears the tachyzoites but not the encysted bradyzoites. This persistence of bradyzoites results in a chronic, lifelong infection that allows further dispersal of Toxoplasma. Thus, parasite differentiation r...

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).…”

“…We previously reported that a PUS is necessary for differentiation of the parasite Toxoplasma gondii (Anderson et al 2009). This single-celled eukaryote can exist within its human host as fast-growing tachyzoites or as slower-growing encysted bradyzoites.…”

“…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.…”

Type II Toxoplasma gondiiKU80Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection

“…Disruption of the SRS9 gene or the SAG2CDXY gene locus revealed a phenotype of normal cyst burdens at 4 weeks postinfection, and then reduced cyst burdens were observed later during latent infection (41,55). Disruption of a pseudouridine synthase homologue (PUS1) revealed a phenotype of slightly smaller cyst size and slightly increased cyst burdens (1). Targeted disruption of the heat shock protein BAG1 gene (6,69) resulted in a minor defect in cyst development.…”

“…Nine distinct targeted gene deletions (1,6,13,32,38,54,55,57,65,69) have been successfully developed in type II strains since genetic transformation of T. gondii was reported nearly 20 years ago (18,39,61). In contrast, recently reported type I KU80 knockout strains that exhibit highly efficient gene replacement frequencies has significantly accelerated the development of targeted gene deletions (2,3,15,20,23,26,34,37,44).…”