Functions And Expression Regulation Mechanisms Of PGK1,GAPDH2 And ENO2 In Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasite,which can infect almost all warm-blooded animals and humans.However,currently no ideal vaccine has been developed to prevent T.gondii infection.Glycolysis is an important metabolic pathway in all cells.In Toxoplasma,many glycolytic enzymes have two isoforms,which have different cellular localization patterns,or show expression differences at different stages during the life cycle.Yet,the biological functions of these different isoforms,as well as the regulation mechanism of their expression in T.gondii,are largely unknown at this point.Focusing on these questions,this study focused on phosphoglycerate kinase(PGK1)localized in the cytoplasm and glyceraldehyde 3-phosphate dehydrogenase 2(GAPDH2)localized in the apicoplast to study the function of glycolytic enzymes with different subcellular localizations.Enolase 2(ENO2),which is specifically expressed in the tachyzoite stage,is selected as the object to study the regulation mechanism of its stage-specific expression.The main results include the following:1.The function of PGK1 in cytoplasm during the growth and metabolism of T.gondiiPGK catalyzes the conversion of 1,3-diphosphoglycerate(1,3-BPG)to3-phosphoglycerate(3-PG),and at the same time generates a molecule of ATP.T.gondii encodes two PGKs,including PGK1 which is located in the cytoplasm and PGK2 in the apicoplast.However their biological functions in the parasites are not clear.In this study,using CRISPR/Cas9-assisted homologous recombination,the promoter of the PGK1 gene was replaced with a promoter which is regulatable by anhydrotetracycline,thereby constructing a conditional knockdown strain iPGK1 in the TATi background.The in vitro phenotyping experiments showed that suppressing the expression of PGK1 reduced the growth of T.gondii under normal culture conditions.However,if the glucose was removed from the culture medium,the growth defect of the PGK1 knockdown strain was largely restored.These results suggest that knocking down PGK1 may cause accumulation of toxic metabolites during glucose metabolism and then inhibit the growth of parasites.Together these data indicate that PGK1 plays a key role during the metabolism of the main carbon source glucose in T.gondii,as such PGK1 is essential for the growth of the parasite.2.Study on the function of GAPDH2 in apicoplastSimilar to PGK,T.gondii also encodes two GAPDHs,of which GAPDH2 is located in the apicoplast.But its role in apicoplast metabolism and T.gondii growth is unclear.We tried to analyze the function of the GAPDH2 gene by genetic modification.The first attempt was to directly knock out GAPDH2,but it’s failed to get a positive strain,suggesting that GAPDH2 may be critical to the growth of T.gondii.Next,we placed the endogenous GAPDH2 gene between two LoxP sites in the DiCre strain,and tried to induce the knockout of GAPDH2 by Cre-mediated recombination.We successfully obtained the DiCre/LoxP-GAPDH2 strain,but for unknown reasons,Cre recombinase could not remove GAPDH2 between LoxP sites.We suspected that the special chromosomal structure of the GAPDH2 gene locus hindered the working efficiency of Cre,so we tried to insert LoxP-GAPDH2 into the UPRT locus first,and then knock out the endogenous GAPDH2 gene.We have also successfully obtained the DiCre/uprt::LoxP-GAPDH2 strain,and the GAPDH2 between the LoxP sites of this strain can be excised by Cre,but we still could not knock out the endogenous GAPDH2.Then we tried the third strategy,adding an AID(Auxin Induced Degradation)tag to the3’ end of the endogenous GAPDH2 gene,and knocking it down by adding auxin to induce the degradation of GAPDH2.We successfully constructed the Tir1/GAPDH2-AID strain,but auxin could not induce the degradation of GAPDH2.It may be because the protein is located in the apicoplast and is not easily degraded by the proteasome in the cytoplasm.Therefore,despite we tried various attempts,currently no strain that can manipulate the expression of GAPDH2 was achieved.It is likely due to the special structure of the GAPDH2 gene locus and its apicoplast localization,which also suggests the limitations of the current conditional genetic modification systems in T.gondii.3.Regulation of G4 chain on ENO2 specific expression in tachyzoitesThere are also two isoforms of ENO in T.gondii: ENO1,which is highly expressed in the bradyzoites and ENO2,which is highly expressed in the tachyzoites.However,the regulatory mechanisms that control their stage specific expression is unclear.We found that the 5’UTR region of ENO2 contains a typical potential G-quadruplex sequence(PQS).It was verified by circular dichroism that the PQS could indeed form a G-quadruplex,suggesting that it may be involved in the regulation of ENO2 expression.At the same time,we used G4 Hunter prediction software to predict all the PQS in the genome of T.gondii,and analyzed the distribution of these PQS in the genome by bioinformatics methods.Among the genes containing PQS,44.37% of them were up-regulated during the tachyzoite stage;51.69% of them are up-regulated in the merozoite stage.Further analysis revealed that almost all glycolytic enzymes contain PQS.As such,we hypothesize that G-quadruplex may be involved in regulating the expression of glycolytic enzymes like ENO2.In summary,this study selected three representative enzymes PGK1,GAPDH2 and ENO2 to analyze the functions and expression regulation mechanisms of glycolytic enzymes in T.gondii.It is verified that PGK1 plays a key role in the glucose metabolism of T.gondii,and it is important for the growth of the parasite.We have tried different approaches to genetically modify the GAPDH2 gene to study its function,but due to limitations of current tools none of them generated a strain that could be used for GAPDH2 functional analysis.We also analyzed the PQS in the whole genome of T.gondii and predicted the potential role in regulating the stage specific expression of glycolytic enzymes like ENO2.