Disruption of a Locus Encoding a Nucleolar Zinc Finger Protein Decreases Tachyzoite-to-Bradyzoite Differentiation in Toxoplasma gondii

Abstract

During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd ؊ ) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd ؊ phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.

A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation inToxoplasma gondii

“…Clone A7 of the Pru strain of Toxoplasma was used as the parental strain in this work (38). A7 lacks the gene for hypoxanthine-xanthine-guanine phosphoribosyl transferase (HPT) (and is thus designated ⌬hpt) and was previously engineered to constitutively express green fluorescent protein (GFP) and firefly luciferase.…”

“…Insertional mutagenesis and reverse genetics have proven successful in creating Tbd Ϫ mutants with identifiable genetic disruptions. Disruption of genes encoding either zinc finger protein 1 (ZFP1) or plasma membrane ATPase 1 (PMA1) results in a 50% decrease in differentiation efficiency in vitro (18,38). Neither of these mutants, however, has provided insight into the molecular mechanisms of T. gondii differentiation.…”

“…EUKARYOT. CELL ated by insertional mutagenesis of Pru strain Toxoplasma, has a differentiation efficiency of ϳ10% relative to WT parasites, making it one of the strongest differentiation mutants of Toxoplasma yet described (38). Multiple attempts at plasmid rescue from this mutant have revealed only a single disruption with multiple, tandem copies of the mutagenesis vector inserted into the same ORF as in TBD8, i.e., 20.m03736.…”

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

“…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).…”

What new cell biology findings could bring to therapeutics: is it time for a phenome-project in Toxoplasma gondii?

“…However, in this approach only non-essential genes for tachyzoite growth in vitro can be identified. Hence this strategy has been mainly employed to identify genes required for differentiation from tachyzoites to bradyzoites (Matrajt et al 2002, Vanchinathan et al 2005). In the future, a combination of ectopic gene regulation systems with random insertional mutagenesis might be possible.…”