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Abstract
Toxoplasma gondiis chronic infection is critical for the parasites persistence and unfortunately, this aspect of the parasite is vastly understudied. This is due to the challenges of culturing the cyst forming bradyzoites in vitro as they are often outcompeted by the rapidly growing tachyzoite form. To overcome this challenge, we generated an Me49 Type II strain containing a plasmid allowing the suppression of tachyzoite growth, selection for bradyzoites, and verification of conversion by stage specific fluorescent protein expression. Optimization of conversion using the cyclic-GMP protein kinase-inhibiting Compound 1 provided mature cysts in vitro to study bradyzoite biology. Addition of the calcium ionophore Ionomycin showed an inability by bradyzoites to egress from mature cysts and mechanical lysis showed an additional inability to re-invade new host cells. Treatment with conditions that mimic the gastrointestinal tract, acidic pH and pepsin protease (Acid/Pepsin) or the pancreatic protease Trypsin, activated bradyzoite invasion with the latter being the most effective activator. Chemical mutagenesis with ENU combined with an enrichment protocol involving mechanical lysis and outgrowth of mutants that no longer required protease treatment allowed the isolation of a parasite population, along with clones, that via whole genome sequencing would allow the identification of genesinvolved in this process. The ability to study bradyzoite biology in vitro provides a unique opportunity to observe and interrogate unexplored aspects of this crucial form of the parasite.