Current approaches to find therapeutic solutions to treat and prevent reactivation of toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. The loss of developmental competency in vitro that is exacerbated during the generation of transgenic tachyzoites is also a major impediment to understanding the molecular basis of bradyzoite recrudescence, which is the central feature of reactivation. We have developed an innovative ex vivo model of bradyzoite recrudescence and applied this method to successfully modify Toxoplasma genes while avoiding the problems caused by continuous in vitro cell culture. We present four protocols required to engineer in vivo transgenic tissue cysts: 1) the reliable production of in vivo tissue cysts and excysted bradyzoites, 2) the use of fast-growing parasites from ex vivo bradyzoite infections to successfully generate transgenic tissue cysts in mice, 3) the cloning of transgenic bradyzoites via single cyst infections, and finally, 4) the long term cold storage and recovery of transgenic tissue cysts in brain tissue homogenates. We demonstrated these protocols by knocking out the Toxoplasma TgHXGPRT gene and the gene encoding the ApiAP2 transcription factor, AP2IX-9 in tissue cysts from mice. Unexpectedly, the knockout of the AP2IX-9 gene in the Type II ME49EW strain eliminated one of the three developmental pathways initiated by the bradyzoite; host-dependent bradyzoite-to-bradyzoite replication.

No abstract