Single cell RNA sequencing reveals unexpected heterogeneity and identifies AP2XI-6 as an in vivo bradyzoite specific cell cycle regulator

Toxoplasma gondii prevalence is due, in part, to its ability to persist in hosts while retaining the capacity to transmit and recrudesce. This process, while critical, is poorly understood. Through single cell RNA profiling of in vivo-derived bradyzoites, we discovered that bradyzoites are heterogeneous and not G1-arrested, as expected from in vitro studies. Instead, they progress through two distinct cell cycles that branch from a single G1 state. One is common amongst tachyzoites and in vitro- and in vivo-derived bradyzoites; the other is unique to in vivo-derived bradyzoites. While in G1b, these in vivo-derived bradyzoites express cyst wall proteins predicted to replenish the wall that protects cysts and prevents clearance. We demonstrated that AP2XI-6, a putative AP2-transcription factor expressed in G1 in vivo, is dispensable for tachyzoite growth and cyst formation in vitro but necessary for encystation in vivo. We propose that AP2XI-6 is therefore a driver of replication through the newly identified in vivo bradyzoite specific cell cycle and that this is required for cyst maturation and persistence.