AP2XII‐9 is essential for parasite growth and suppresses bradyzoite differentiation in <i>Toxoplasma gondii</i>

Cyst formation, resulting from the differentiation of rapidly replicating tachyzoites into slowly growing bradyzoites, is the primary cause of chronic toxoplasmosis. Although the mechanisms governing bradyzoite differentiation have been partially elucidated, they remain incompletely understood. In this study, we show that the transcription factor AP2XII-9 is localized in the nucleus and exhibits periodic expression during the tachyzoite stage, with peak expression observed during the synthesis and mitosis phases. Conditional knockdown of AP2XII-9 in both the type I RH strain and type II cyst-forming Pru strain revealed that AP2XII-9 plays a critical role in the lytic cycle by regulating the formation of the inner membrane complex, proper apicoplast inheritance, and normal cell division, underscoring its essential role in T. gondii growth. Furthermore, depletion of AP2XII-9 induced bradyzoite differentiation even in the absence of alkaline stress. Transcriptomic analysis revealed that the deletion of AP2XII-9 resulted in the downregulation of tachyzoite growth-related genes and upregulation of a series of bradyzoite-specific genes. Taken together, these findings indicate that AP2XII-9 is essential for maintaining the rapid and normal replication of tachyzoites while actively repressing bradyzoite differentiation, reflecting the complexity of the mechanisms underlying bradyzoite differentiation.