AP2X-1 is a negative regulator of <i>Toxoplasma gondii</i> sexual commitment

<i>Toxoplasma gondii</i> is a widespread protozoan with a complex life cycle, characterized by transitions between various hosts and developmental stages, each tailored to a specific niche within its host. However, the regulatory mechanisms governing these life cycle transitions are not well understood. In this study, we investigated the AP2 factor AP2X-1, which is expressed during the tachyzoite and bradyzoite stages but decreases in the mature merozoite stage. Knockout of <i>ap2X-1</i> significantly impaired tachyzoite invasion and replication while increasing the frequency of bradyzoite differentiation. As a component associated with the HDAC3/MORC complex, loss of <i>ap2X-1</i> led to the upregulation of bradyzoite- and sexual stage-specific genes. Single-cell sequencing revealed that the <i>ap2X-1</i> knockout strain exhibited a mixed population of tachyzoite-, bradyzoite-, merozoite-, and sporozoite-like parasites. Cleavage under targets and tagmentation analysis revealed a substantial overlap between AP2X-1 and the HDAC3/MORC complex at the promoters of bradyzoite- and sexual stage-specific genes. Additionally, assay for transposase-accessible chromatin with high-throughput sequencing analysis demonstrated that AP2X-1 influences chromatin compaction and accessibility, suggesting that AP2X-1 may modulate the function of the HDAC3/MORC complex to facilitate the repression of bradyzoite differentiation and sexual commitment. Loss of <i>ap2X-1</i> resulted in significant attenuation of <i>T. gondii</i> virulence and decreased brain cyst formation <i>in vivo</i>. These findings identify AP2X-1 as a critical negative regulator of <i>T. gondii</i> sexual development.IMPORTANCE<i>Toxoplasma gondii</i> undergoes a complex life cycle characterized by alternating developmental stages. The genetic reprogramming mechanisms driving these stage transitions remain largely unknown. In this study, we identified the AP2 factor AP2X-1 as a critical regulator important for <i>T. gondii</i> growth and life cycle progression. Our findings suggest that AP2X-1 functions as a repressor by modulating the function or influencing the association of the HDAC3/MORC complex at the promoters of bradyzoite- and sexual stage-specific genes, leading to chromatin compaction, restricting DNA accessibility and thereby repressing the transcription of genes required for bradyzoite formation and sexual commitment. Deletion of <i>ap2X-1</i> significantly reduced <i>T. gondii</i> virulence and its ability to form brain cysts. These findings reveal a previously unknown regulatory pathway controlling sexual development in <i>T. gondii</i>, providing new insights into its underlying mechanisms.