Regulation Of Toxoplasma Gondii Growth And Development By AP2X-4 And AP2X?-5

Toxoplasma gondii is an obligate intracellular parasite that infects all warmblooded animals,including humans.It can lead to serious toxoplasmosis such as abortion and still birth.T.gondii has a complex life cycle,including the sexual stage in the definitive host and the asexual stage in intermediate hosts.The asexual stage consists of the tachyzoite stage with rapid replication of parasites during acute infection and the bradyzoite stage with slowly-growing parasites during chronic infection.Different stages of the parasites have different biological characteristics,which play key roles in the transmission and pathogenesis of Toxoplasma.But we know little about the regulation mechanisms that control the differentiation of the parasites from one stage to another.Two transcription factors(AP2X-4 and AP2X?-5)with different abundance in tachyzoites and bradyzoites were identified by nuclear proteomics in our laboratory previously.This study focused on the functions of these two proteins to understand their roles in regulating parasite growth and development.The main work includes the following:(1)AP2X?-5 regulates gene expression in different stages of the life cycleIn the previous study,a mutant strain lacking AP2X?-5 has been constructed in the type II strain ME49.In this study,a specific antibody for AP2X?-5 was obtained and it was used to validate the successful deletion of AP2X?-5 in the mutant strain.RNA-seq analysis found that the deletion of AP2X?-5 leads to alteration of the expression of genes that are stage regulated,in particular,the upregulation of genes in tachyzoites that are normally only expressed during sexual stages.Such disorder of gene expression leads to the decrease of virulence and the reduced bradyzoite growth.DNA-binding assay showed that a recombinant protein containing the AP2 domain of AP2X?-5 was able to specifically bind the LDH2 promoter.These results indicate that the AP2X?-5 transcription factor regulates gene expression in each stage of the life cycle by binding to the promoters of target genes,thus influencing the virulence of the parasites.(2)AP2X-4 regulates the expression of rhoptry proteinsThe AP2X-4 transcript level was higher in bradyzoites than that in tachyzoites.Therefore we suspected that it may regulate bradyzoite differentiation.However,genetic studies have shown that it is important for the growth of tachyzoites.We found that knocking out AP2X-4 in the type II strain ME49 severely impaired parasite invasion and growth.RNA-seq analyses demonstrated that the expression of a number of rhoptry proteins,such as RON4 and RON5 that are key for parasite invasion,were decreased.In addition,AP2X-4 also regulates a subset of genes that are specifically expressed during chronic infection,such as BAG1 and LDH2.Given the diverse targets of AP2X-4,its inactivation led to drastic attenuation of parasite virulence and complete inability to form chronic infection.Toxoplasma gondii encodes 67 AP2 transcription factors.In this study,we focused on AP2X-4 and AP2X?-5 and found that AP2 factors have diverse roles in regulating activities in the parasites.Different factors have different regulatory targets.AP2X?-5 represses the expression of genes in tachyzoites that are normally expressed in other stages of life cycle,to maintain virulence of the parasite.AP2X-4 regulates parasite invasion of host cells by controlling the expression of rhoptry proteins,thereby affecting the growth and virulence of the parasite.