Functions And Regulation Mechanisms Of DnaK-TPR And LDH2 Gene During Toxoplasma Gondii Growth And Development

Toxoplasma gondii is an obligate intracellular and opportunistic protozoan.It is capable of infecting virtually all warm-blooded animals including humans,and has a world-wide distribution,causing serious toxoplasmic encephalitis,ophthalmopathy,abortion and fetus death in susceptible individuals and animals.There are two stages in intermediate hosts,acute infection and chronic infection.During the infection of a na?ve host,T.gondii first quickly multiplies as tachyzoite,but in heathy host,as a response to host immune clearance,some of the tachyzoites can convert to the slowly replicating bradyzoites and stay within host cells in the form of tissue cysts,causing chronic infection and stay with the hosts lifelong.Currently there are no effective treatments for chronic toxoplasmosis.However,when the host immune system is compromised,bradyzoites in the tissue cysts may get reactivated and convert to tachyzoites,leading to acute infection again and causing host damage of organism and even death.The interconversion between tachyzoites and bradyzoties plays an critical role in the transmission and pathogenesis of T.gondii.To understand the biology and mechanisms underlying bradyzoites development will help us illuminate the molecular basis of T.gondii growth and development,in order to provide theoretical basis for effective T.gondii drug and vaccine design.In this study,we characterized two genes that are dramatically up-regulated during bradyzoite,tetratricopeptide repeat(TPR)structural motif containing protein DnaK-TPR and lactate dehydrogenase LDH2.We sought to identify the functions and regulation mechanisms of DnaK-TPR and LDH2 genes,to analyze their roles during T.gondii growth and development.Through CRISPR/CAS9 directed gene editing technology,we were knocked out DnaK-TPR for functional analysis.We were also identified the cis regulatory elements and transcription factors in LDH2 promoter,in order to explore its molecular regulation mechanism during bradyzoite differentiation.Finally,we were found a novel transcription factor and researched its function in T.gondii by gene knockout.The main work of this dissertation including the following aspects:(1)Construction of the DnaK-TPR knockout strain and phenotypic analysisDisruption of a protein that contain tetratricopeptide repeat(TPR)structural motifs named DnaK-TPR,which is up-regulated in the bradyzoite stage using CRISPR/CAS9 system.After obtained DnaK-TPR mutant,we demonstrated that DnaK-TPR didn't affect the growth of tachyzoites in vitro by plaque assay.Replication assay indicated thatDnaK-TPR displayed very similar replication dynamics as the parental strains,suggesting that DnaK-TPR do not play critical roles for tachyzoite replication in vitro.Bradyzoite differentiation assay revealed that DnaK-TPR deletion mutant had similar bradyzoite formation efficiencies as the parental strain,suggesting that this gene did not have significant roles in bradyzoite formation in vitro.Parasite virulence in mice suggesting that the virulence of DnaK-TPR mutant was slightly attenuated in laboratory mice.Cyst counting suggesting that there was no significant difference in brain cyst formation between the DnaK-TPR knockout mutant and parental strain.So we consider that although DnaK-TPR expression was significantly up-regulated during bradyziote formation,it may be a non-intentional target in the control mechanism of bradyziote development.It can response to bradyzoite differentiation regulatory mechanism,but not directly involved in the bradyzoite formation.(2)Research on the regulation mechanism of LDH2 gene specifically expressed at the bradyziote stageDetect the differential activation of the LDH2 promoters during tachyzoite and bradyzoite by deletion analysis of 5'-flanking regions fused to the EGFP reporter followed by transient transfection.We determined the core region of LDH2 promoter,and confirmed a GTGTGT repeat motif was a key regulatory element in LDH2 promoter.Co-Ip assay demonstrated AP2XI-4 transcription factor can specifically bound to LDH2 promoter,suggesting that AP2XI-4 was transcription factor involved in LDH2 gene expressing during bradyzoite.In order to find more genes involved in bradyzoite development,we analyzed the differences between tachyzoite nucleoprotein and bradyzoite nucleoprotein by proteomics,found a AP2?-5 transcription factor that was up-regulated during bradyzoite formation.We knocked out AP2?-5 gene by CRISPR/CAS9 technology,using AP2?-5mutant for phenotypic analysis.We demonstrated that disruption of AP2?-5 didn't affect the growth of tachyzoites and bradyzoite differentiation in vitro,also had no significant difference in brain cyst formation between the AP2?-5 knockout mutant and parental strain.However,the virulence of the knockout strain was significantly reduced,which could not lead to the death of infected mice.Quantitative real-time PCR results displayed that in AP2?-5 mutant,BAG1,LDH2 and ENO1 m RNA level were down-regulated during bradyzoite,suggesting that AP2?-5 is a transcription factor involved in regulating T.gondii virulence,and play a key role in regulating LDH2 geneexpression.