Mechanisms regulating reactivation pathways of Toxoplasma gondii as revealed by bradyzoite transgenesis

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 parasite stage of reactivation. We have successfully modified Toxoplasma genes in the bradyzoite tissue cyst stage using ex vivo bradyzoite-based methods. Our new protocols validated the clonality of individual cysts and confirmed that single tissue cysts can robustly infect mice. We demonstrated these protocols by generating in vivo bradyzoites carrying a knockout of the Toxoplasma HXGPRT gene or the gene encoding the ApiAP2 transcription factor, AP2IX-9. 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. Our genetic protocols were further validated by producing in vivo bradyzoites lacking the bradyzoite-specific cyclin, TgCYC5. Interestingly, genetic ablation of TgCYC5 led to a large number of small cysts that formed from single mother cysts in mouse brain. Further study revealed the cause of the small-cyst phenotype in TgCYC5 knockout parasites was a disruption in the normal balance of bradyzoite subtypes that enhanced the bradyzoite-to-bradyzoite developmental pathway. These new data demonstrate the feasibility of generating transgenic parasites in a developmentally competent strain using ex vivo bradyzoite-based methods. Furthermore, by targeting the AP2IX-9 and TgCYC5 genes, whose transcripts are upregulated specifically in bradyzoites, we have clarified how these factors influence the pathways of tissue cyst recrudescence.