The Role Of Bromodomains In The Lytic Cycle Of Toxoplasma Gondii

Toxoplasma gondii is the organism responsible for the lethal disease of Toxoplasmosis,which has a tremendous impact on humans and most warm-blooded animals,especially for immunocompromised individuals and pregnant women.The life cycle of T.gondii is complex,these life action all rely on fine-tune genes expression by the epigenetic control mechanisms to facilitate different hosts/tissues or environment stress.The lytic cycle can be divided into three stages,invasion to the cell,replication in the cell,and egress from the host cell.Those can ensure the survival of the parasites.Bromodomains(BRDs)is a highly conserved structural module domain.This domain is found in various proteins,including chromatin,nuclear acetyltransferases,and transcription-associated proteins.In this study,TgBDPs act as the research object to explore the regulation of the growth of the T.gondii,mainly including the following four parts.Firstly,to address the role TgBDPs plays in the context of acute infection,we attempted to use the clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 and an auxin-inducible degron-based conditional knockout strategy.In this study,the physicochemical properties and structure of T.gondii bromodomain-containing proteins were identified by bioinformatics analysis.Then used CRISPR/Cas9 technology to insert the Sm FP-HA tag endogenously into the C-terminus of TgBDPs,and,through western blot and polymerase chain reaction,we successfully constructed a conditional knockout strain TgBDP4-Sm FP-HA and TgBDP5-Sm FP-HA.Our data releveled that both TgBDP4 and TgBDP5 localized in the parasite’s nucleus and remained unchanged during the parasite’s replication.Conditional ablation of TgBDP4 and TgBDP5 through an auxin-inducible degron-based knockdown strategy caused a growth defect in parasite replication.Notably,TgBDP4 is required and essential genes for the growth of the parasite.Secondly,our transcriptome data releveled that depletion of the TgBDP5 led to changes in the expression level of 847 genes,in which 392 genes down-regulated and482 genes up-regulated.And seven AP2 transcription factors(TF)altered arouse our attention.The transcriptome of TgBDP5-deficient parasites was analyzed to investigate the regulating role of this protein.The expression level of 179 genes altered upon the depletion of TgBDP5,in which 156 were up-regulated and 23 was down-regulated.The results indicated that TgBDP4 and TgBDP5 DEGs related to cellular processes and metabolism pathways.The associated secondary pathways included amino acid metabolism,carbohydrate metabolism,cell growth and death,cellular community-eukaryotes.T.gondii provides energy and nutrients for itself through the metabolic and synthetic processes involved in these DEGs to maintain its own growth and development.Then the Tg AP2X-5 interacted with Tg AP2XI-5,they can regulation of virulence genes in T.gondii.The result in virulence factors ROP37,ROP15,ROP30,MIC3 and MIC12 down-regulate,ROP18,ROP20,ROP5 A and ROP5 B up-regulated,were verified by q RT-PCR analysis.As a conserved BRD-containing protein,TgBDP4 may act as a potential transcriptional regulator.Deletion of TgBDP4 result in Tg AP2X-5 down-regulate,Tg AP2X-5 expression leads to defects in Tg AP2XI-5binding to multiple crucial virulence factors gene promoters.Eventually affect replication and lead to lethal defect.As a unique hypothetical bromodomain-containing protein,the two conserved amino acids used for binding the acetylated-lysine are missing in the TgBDP5.Therefore,TgBDP5 may not "read" the Kac.Finally,using the bioinformatics analysis to predict the interacting proteins of TgBDP4 protein,eight interacting proteins were obtained.Co-immunoprecipitation was used to explore the interaction.The results showed that four proteins eukaryotic expression vectors were successfully constructed,but the interaction has not been verified yet.In order to explore the effect of TgBDP5 deletion of mice,virulence experiments showed that deletion of TgBDP5 reduced the virulence of mice and improved the survival rate,indicating that TgBDP5 plays an important role in the growth and pathogenicity of T.gondii.In summary,we used CRISPR/Cas9 technology,AID conditional knockout strategy,transcriptomics,to estimate the regulatory mechanisms of the TgBDP4 and TgBDP5 in the lytic cycle of parasite.Our data releveled that TgBDP4 and TgBDP5 caused a growth defect in parasite replication.DEGs related to cellular processes and metabolism pathways,demonstrate that those DEGs play an important role in the growth of the parasite.As a conserved domain,BRD act as a vital function of T.gondii.It may act as an important candidate for the drug targets of the parasite.