Construction Of Knockout Toxoplasma Gondii Strains Of Key Amylopectin Metabolic Enzymes And Their Basic Biological Characteristics

Toxoplasma gondii is an obligate intracellular parasite that causes severe disease in immunocompromised patients.Two forms of T.gondii,the tachyzoites in the acute stage and the bradyzoites in the chronic stage,are presented in the intermediate host.In immunocompromised hosts,the latent infection of T.gondii can be reactivated from the tissue cysts.The presence of a large amount of amylopectin in cysts ensures the better survival of T.gondii in the absence of nutrients.Many enzymes are involved in amylopectin metabolism,among them,4aGT and Aa16 GL play important roles in the catabolism process,and GT8 play important roles in the anabolism process.In this study,the CRISPR-Cas9 mediated homologous terminal conjugation technique was used to insert 3×HA tag proteins at the C ends of 4aGT,Aa16 GL and GT8 genes.The HA fused protein is expressed by endogenous promoters.The IFA was used to observe the subcellular localization of the three proteins in tachyzoites.However,no localized fluorescence was observed in 4aGT and GT8 by IFA using HA antibody.In addition,we constructed recombinant plasmids p ET30a-4aGT and p ET30a-GT8,which were transformed into BL21 competent cells for prokaryotic expression after ITPG induction.The SDS-PAGE electrophoresis results showed that the size of 4aGT protein was about 70 KDa,and that of GT8 protein was 43.2 KDa,which were consistent with the expected sizes of proteins.The results of protein electrophoresis after ultrasonic fragmentation showed that both 4aGT and GT8 proteins were expressed in the form of inclusion bodies,and the polyclonal antibodies of these two proteins were obtained by immunizing rabbits.However,the localization of these two proteins by IFA using the rabbits anti-4aGT or GT8 also failed to be observed.Using the CRISPR-Cas9 mediated homologous recombination technique,4aGT,Aa16 GL and GT8 gene knockout strains of T.gondii type I RH strain and PRU type II strain were constructed.The results showed that the knockout of 4aGT and GT8 genes had no effect on T.gondii tachyzoite egress,plaques,intracellular replication,virulence assay in mice,bradyzoite differentiation or brain cysts assay.The knockout of Aa16 GL gene had no effect on tachyzoite egress,intracellular replication,bradyzoite differentiation and virulence assay in mice.However,the knockout of the Aa16 GL gene made the plaques get smaller and less,a slight growth defect,and a reduced number of brain cysts in the mice.Deletion of CDPK2 or GP can lead to abnormal accumulation of amyloid in T.gondii,Aa16 GL can be phosphorylated by CDPK2 as amyloid metabolic enzymes.Therefore,transmission electron microscopy was used to observe whether the 4aGT,Aa16 GL and GT8 gene knockout strains of PRU strains affect the metabolism of amylopectin in tachyzoites.The results showed that the deletion of 4aGT,Aa16 GL and GT8 genes in T.gondii did not lead to the abnormal accumulation of amylopectin in tachyzoites,and whether it affect the glucose flux and the accumulation of amylopectin in other periods of T.gondii remain to be further studied.In summary,we constructed 4aGT,Aa16 GL and GT8 knockout strains in T.gondii RH and PRU strains.The basic biological functions of 4aGT,Aa16 GL and GT8 were elucidated.We found that the deletion of Aa16 GL gene caused the smallar sizes and smaller numbers of plaques and reduced number of cysts in mice.These results not only lay the foundation for further studies on other biological functions of amylopectin metabolic enzymes in T.gondii,but also provide reference for research on effective anti-Toxoplasma gondii drugs or vaccines.