The Regulatory Role And Mechanism Of TgAP2XI-3 On Tachyzoite Growth And Cell Cycle In Toxoplasma Gondii

Toxoplasma gondii is a very important intracellular protozoan with a wide range of hosts and a complex life cycle,causing serious zoonotic parasitic diseases.The life activities of T.gondii,such as DNA anabolism,RNA anabolism,protein anabolism and cell division,are strictly regulated.APi AP2 family of transcription factors are an important part of the transcriptional regulatory network of T.gondii and has been shown to be involved in the regulation of important growth and development processes such as cell cycle and stage transition.In the development of a targeted drug for toxoplasmosis,a short-strand DNA analogues(phosphorylamides morph oligomers)targeted TgAP2XI-3 to inhibit its expression and significantly inhibit the growth of T.gondii tachyzoites,showing promising therapeutic potential.However,the biological function of TgAP2XI-3 is not determined.Therefore,this study focuses on the transcription factor TgAP2XI-3,by using reverse genetics to analyze the role and mechanism of this transcription factor in the growth development of parasite and transcriptional regulation.Related research methods and results are listed as follows:1.TgAP2XI-3 is critical for the growth of toxoplasma tachyzoites.The conditional deletion strain of TgAP2XI-3 was constructed using the mini-AID system.Exogenous addition of IAA leads to the completely degradation of AP2XI-3 protein.The degradation of TgAP2XI-3 protein did not affect the invasion ability of T.gondii,but the replication ability was severely surpressed,unable to form visual spots,and lead to a significant decrease in the virulence of the missing strain in mice.Above results suggests that TgAP2XI-3 is essential to the growth of Toxoplasma gondii in vitro and in vivo.2.TgAP2XI-3 plays an important role in the cell cycle of T.gondii tachyzoite.The cell cycle was a important factor in determining its replication rate,and the degradation of TgAP2XI-3 resulted in severe arrest of T.gondi replication.Therefore,we examined cell cycle progression of T.gondii tachyzoite before and after TgAP2XI-3degradation.The results showed that the G1 phase of T.gondii was significantly arrested after degradation of TgAP2XI-3,indicating that the regulation role of T.gondii AP2XI-3 is very critical for the G1 phase of T.gondii cell cycle.3.TgAP2XI-3 regulates the key enzymes and proteins that regulate the synthesis of macromolecules such as RNA and protein in T.gondii,and is an important factor in regulating the G1 phase of T.gondii cell cycle.Transcription and Chip-seq results showed that TgAP2XI-3 targeted and regulated the transcriptional expression of a large number of RNA polymerases,RNA cleavages,helicases,ribosomal proteins,translation initiation factors and other genes,indicating that TgAP2XI-3 is a key regulator of the synthesis of m RNA and protein and other macromolecules of T.gondii.TgAP2XI-3 deletion caused a severe arrest in the G1 phase of Toxoplasma gondii,which is primarily responsible for initiating a new round of transcription,restoring the ability to synthesize large amounts of proteins,and establishing a new round of DNA replication system.Therefore,we combined TgAP2XI-3transcriptome and Chip-Seq results with a gene population highly expressed in the cell cycle dependent G1 phase of T.gondii.The results showed that TgAP2XI-3 targets a large number of genes highly expressed in G1 phase,and these targeted genes are mainly highly expressed in the early G1 phase(G1a),suggesting that TgAP2XI-3 regulates the early G1 phase of the toxoplasma cell cycle.In summary,this study explored the regulatory role and molecular mechanism of the transcription factor TgAP2XI-3 on T.gondii growth and cell cycle by using reverse genetics approach,transcriptomics and Chip-seq.The results revealed that the transcription factor TgAPXI-3,which is essential for T.gondii growth,is a master regulator of the G1 phase of the T.gondii cell cycle and a rate-limiting regulator of T.gondii m RNA,protein and other macromolecular synthesis.This study further elucidates the tachyzoite cell cycle of T.gondii and its regulatory mechanism,providing theoretical basis for TgAP2XI-3 as a candidate drug target for treatment of toxoplasmosis.