A Genetic Screen Identifies Factors Required for Toxoplasma Differentiation into Latent Forms

Toxoplasma differentiates from acute stage tachyzoites into latent bradyzoite forms in the muscle and central nervous system. How Toxoplasma senses the tissue environment and undergoes differentiation, at the molecular level, in poorly understood. We undertook a whole genome CRISPR screen in RH strain to determine genes required for utilization of different carbon sources and serendipitously discovered that switching to glutamine, from glucose, triggers a differentiation-like program, highlighting 60 genes involved in this process. We show that glutamine, as the sole carbon source, also triggers differentiation in cytogenic strains and relies on the transcription factor BFD1. We then generated a CRISPR sub-library of these 60 genes and tested their role in differentiation in cystogenic strains in vitro and in mice. Individual mutants show that several genes involved in central carbon metabolism are required for differentiation in vitro and in vivo as well as genes involved in regulation of RNA. We also identified orthologs of the "GID" E3 ubiquitin ligase complex, which regulates translational repression in during embryogenesis in Drosophila. By genetic tagging and immunoprecipitation, we show that Toxoplasma also has a GID complex, whilst gene knockouts of GID components show impaired differentiation. Current work is centered around identifying TgGID ubiquitinated substrates. This work, therefore, identifies new molecular players and biological processes, including ubiquitination, required for Toxoplasma to differentiate into latent forms