Regulation Of Cell Division Pattern And Expression Of Merozoite Specific Genes By TgAP2Ⅻ-1 In Toxoplasma Gondii

Toxoplasma gondii is an important zoonotic pathogen,which could infects almost all warm blooded animals,such as humans,pigs,cows,birds and so on,and causes severe threat to human health and poultry industry.The life cycle of Toxoplasma gondii is complex and contains multiple developmental stages,which include asexual conversion that exists in immediate hosts,merogony and sexual reproduction that exclusively perform in cat intestine.Oocyst,the final product from sexual reproduction,is a major form in the environmental transmission of Toxoplasma gondii.Therefore,it is very important to elucidate the related mechanisms underlying the sexual developmental conversion of Toxoplasma gondii,which is meaningful for us to understand the life cycle and design effective strategy controlling the transmission of Toxoplasma gondii.But,duo to limited culture strategy in sexual stages of Toxoplasma,the molecular mechanism of merogony and sexual reproduction of Toxoplasma in cat intestine is still poorly understood.Transcriptional regulation plays an important role in the developmental stage transformation of Toxoplasma gondii.Those differentially expressed transcription factors at different developmental stages play an important role in the change of gene expression patterns during the developmental stage transformation.Previous studies have demonstrated that several members of AP2 transcription factor family and the transcription factor BFD1 with the Myb domain control the tissue-cyst formation ability of Toxoplasma gondii in intermediate hosts by regulating bradyzoite developmental genes.However,is transcriptional regulation involved in merogony of Toxoplasma gondii?What are key regulators responsible for merogony in cat intestine?By comparing the differential transcriptomics of several transcript profiles of tachyzoites,bradyzoites and merozoites of Toxoplasma gondii,we found a transcription factor,named AP2Ⅻ-1,is involved in the merogony of Toxoplasma gondii.Then we use the mini-AID knockout system to detailly investigate the biological functions and regulatory mechanisms of AP2Ⅻ-1.The main research results are as follows:1.The division pattern and morphological characteristics of AP2Ⅻ-1 depleted strain are similar with merozoitesIn order to explore the biological function of AP2Ⅻ-1,a conditional knockout strain that can successfully degrade AP2Ⅻ-1 at the protein level was constructed using the miniAID conditional knockout system.Then a series of phenotypic experiments were carried out and we found that AP2Ⅻ-1 depleted strains produce many parasitophorous vacuole(PV)containing abnormal number of offspring,suggesting that the lacking of AP2Ⅻ-1 disrupted the stability of endodyogeny.By using immunofluorescence staining and transmission electron microscopy,we found that AP2Ⅻ-1 depleted strain underwent merogony instead of endodyogeny and produce multinucleated parasites and multiple budding parasites,which both are classic characteristics of merozoites,and finally formed elongated offspring.These phenotypes suggested the important role of AP2Ⅻ-1 in controlling the conversion from tachyzoites to merozoites.Finally,we also investigated the bradyzoite conversion ability of AP2Ⅻ-1 depleted strain and found the PV membrane containing AP2Ⅻ-1 depleted parasites exhibited obvious DBA-positive signal in the normal culture condition(pH=7.4),consistent with the unidirectionality of the stage development whose conversion from tachyzoites to merozoites needs undergo bradyzoites stage firstly.2.The transcript profiles of AP2Ⅻ-1 depleted strains are similar with merozoitesRNA-seq were used to investigate the changes between transcriptomes of AP2Ⅻ-1 depleted strain and wild type.Then we found more than 400 upregulated genes with a 8 fold abundance in AP2Ⅻ-1 depleted strain compared to that in wild type.A categories analysis revealed majority of upregulated genes were merozoite specific and a fraction of them were bradyzoite specific.These upregulated genes contained more than 30 merozoites specific SRS genes,more than 20 toxoplasma Family A,Family B and Family C proteins which is exclusively expressed in merozoites and many classic secreted proteins,such as BRP1,MIC17A,GRA11B and so on.By analyzing those downregulated genes in AP2Ⅻ1 depleted strain,we found more than half of them were also disappear in merozoites,including AMA1,RON2,RON4 and RON5,which is responsible for tachyzoite invasion.In addition,many genes involved in cell division,such as AP2IX-5 and many inner membrane complex proteins(IMCs),were also downregulated in AP2Ⅻ-1 depleted strain.These results suggest that the transcript profiles of AP2Ⅻ-1 depleted strains are similar with merozoites.3.Regulation of merozoite specific genes by AP2Ⅻ-1In order to explore the mechanism of AP2Ⅻ-1 in regulating merogony,chromatin immunoprecipitation(ChIP-seq)and co-immunoprecipitation(Co-IP)were also performed in this study to explore the distribution of AP2Ⅻ-1 on chromosome and the related proteins which interacts with AP2Ⅻ-1.Then we found AP2Ⅻ-1 directly targeted promoters of more than 200 upregulated genes in AP2Ⅻ-1 depleted strain,of which more than 170 were significantly upregulated in merozoites.These results suggest that AP2Ⅻ1 may regulates the expression of merozoite specific genes during the tachyzoite stage.In addition,an important epigenetic factor,MORC,was found a tight interaction with AP2Ⅻ1.MORC/HDAC3 complex has been proved an key role in silencing gene expression through deacetylation of histone H4 on targeted gene loci.The interaction between AP2Ⅻ1 and MORC/HDAC3 complex suggests that AP2Ⅻ-1 may silence merogony related genes in tachyzoite by recruiting MORC/HDAC3 complex.In conclusion,we detailly investigate biological function of AP2Ⅻ-1 using gene editing,transmission electron microscopy,RNA-seq,ChIP-seq and Co-IP and found the key role of AP2Ⅻ-1 in regulating merogony and merogony related genes in tachyzoites of Toxoplasma.These results provide a new insight in the understanding of mechanisms of developmental process of Toxoplasma in cat intestine,and could help us design effective prevention schemes for Toxoplasma transmission from cats.