TgAP2X-7 is a novel cell cycle-regulated transcription factor that plays an essential role in Toxoplasma tachyzoite propagation.

Abstract

Apicomplexan AP2 (ApiAP2) family proteins are a family of transcription factors that are known to regulate gene expression in apicomplexan pathogens, including Toxoplasma. In this study, we focused on TgAP2X-7, a member of the APiAP2 family that is predicted to be essential for Toxoplasma fitness. Endogenous tagging of TgAP2X-7 followed by immunofluorescence analysis revealed that it's a cell cycle-regulated nuclear protein with peak expression in the G1 phase. Since TgAP2X-7 is predicted to be essential for parasite growth, we adopted an auxin-inducible degron (AID) based conditional knockdown approach to understand its function. Phenotypic analysis of the conditional knockdown mutant of TgAP2X-7 showed that the protein is indeed essential for Toxoplasma propagation in vitro, and loss of this transcription factor results in a major defect in invasion and a minor defect in replication. Examination with cell division markers indicated that the absence of TgAP2X-7 results in defects in endodyogeny. Transcriptomic analysis indicated that loss of TgAP2X-7 leads to dysregulation of global gene expression in the parasite, including genes required for host-cell invasion, metabolism, and gene expression. Additionally, Cleavage Under Targets and Tagmentation (CUT&TAG) analysis suggests that TgAP2X-7 likely binds to an 11 bp motif [C/T/G]GCATGCA[G/C/A][C/T/G][G/A] in the parasite genes' promoter region. Together, these findings suggest that TgAP2X-7 is a novel transcriptional regulator in Toxoplasma that governs the expression of genes required for parasite propagation.IMPORTANCEToxoplasma gondii is a protozoan parasite that can cause life-threatening disease in mammals; hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. In this study, we identify and characterize a member of the Apicomplexan AP2 (ApiAP2) family, TgAP2X-7, a developmentally regulated transcription factor. By generating conditional mutant TgAP2X-7, we show that this protein is required for Toxoplasma propagation in vitro, and the absence of this protein results in parasites with significantly reduced competency in invasion, moderate deficiency in replication, and defects in cell division. Importantly, TgAP2X-7-deficient parasites show global changes in gene expression profile, including decreased expression of genes important for Toxoplasma entry into the host cell. Additionally, we identified an 11 bp DNA motif likely recognized by this transcription factor. Hence, this study provides an initial insight into the function of a novel cell cycle-regulated transcription factor essential for Toxoplasma growth.