Mandadi Padmaja, Anandakrishnan Ramu, Gaji Rajshekhar Y
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia, USA.
Department of Biomedical Sciences, Edward Via College of Osteopathic Medicine (VCOM), Blacksburg, Virginia, USA.
mSphere. 2025 Sep 8:e0043825. doi: 10.1128/msphere.00438-25.
Apicomplexan AP2 (ApiAP2) family proteins are a family of transcription factors that are known to regulate gene expression in apicomplexan pathogens, including . In this study, we focused on TgAP2X-7, a member of the APiAP2 family that is predicted to be essential for 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 propagation 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 that governs the expression of genes required for parasite propagation.IMPORTANCE 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 propagation 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 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 growth.
顶复门AP2（ApiAP2）家族蛋白是一类转录因子，已知其可调节顶复门病原体中的基因表达，包括……。在本研究中，我们聚焦于TgAP2X-7，它是APiAP2家族的一员，预计对……的适应性至关重要。对TgAP2X-7进行内源性标记，随后进行免疫荧光分析，结果显示它是一种细胞周期调控的核蛋白，在G1期表达达到峰值。由于预计TgAP2X-7对寄生虫生长至关重要，我们采用了基于生长素诱导降解子（AID）的条件性敲低方法来了解其功能。对TgAP2X-7条件性敲低突变体的表型分析表明，该蛋白确实对……的繁殖至关重要，这种转录因子的缺失会导致侵袭方面的主要缺陷和复制方面的轻微缺陷。用细胞分裂标记物进行检测表明，TgAP2X-7的缺失会导致内二分裂出现缺陷。转录组分析表明，TgAP2X-7的缺失会导致寄生虫体内整体基因表达失调，包括宿主细胞侵袭、代谢和基因表达所需的基因。此外，靶向切割与标记（CUT&TAG）分析表明，TgAP2X-7可能与寄生虫基因启动子区域中的一个11碱基基序[C/T/G]GCATGCA[G/C/A][C/T/G][G/A]结合。总之，这些发现表明TgAP2X-7是……中的一种新型转录调节因子，它控制着寄生虫繁殖所需基因的表达。重要性……是一种原生动物寄生虫，可在哺乳动物中引发危及生命的疾病；因此，确定寄生虫生长和发病机制所需的关键因素对于开发新型治疗方法很重要。在本研究中，我们鉴定并表征了顶复门AP2（ApiAP2）家族的一个成员TgAP2X-7，它是一种发育调控的转录因子。通过构建条件性突变体TgAP2X-7，我们表明该蛋白是……繁殖所必需的，该蛋白的缺失会导致寄生虫在侵袭能力上显著降低，在复制方面有中度缺陷，以及在细胞分裂方面有缺陷。重要的是，缺乏TgAP2X-7的寄生虫在基因表达谱上呈现全局变化，包括对进入宿主细胞很重要的基因表达下降。此外，我们鉴定出了一个可能被这种转录因子识别的11碱基DNA基序。因此，本研究初步揭示了一种对……生长至关重要的新型细胞周期调控转录因子的功能。
