Construction And Phenotypical Analysis Of Three Thioredoxin-deficient Strains Of Toxoplasma Gondii

Toxoplasma gondii is an apicomplexa protozoan which can infect almost all warm-blooded animals,with a worldwide distribution.This parasite can invade virtually all kinds of nucleated cells.T.gondii is an aerobic organism,and its tachyzoite stage requires rapid replication in host cells.Therefore,T.gondii has developed various antioxidant mechanisms to balance its redox state,including the thioredoxin(Trx)system.At present,there are limited researches on the function of Trx family.In this study,three Trx-deficient strains were constructed to explore the effects of the three Trx deletions on the phenotypic function of T.gondii.1.This study utilized the CRISPR-Cas9 technology to knock out two Trx(CTrp26 and CTrx1)genes in the T.gondii RH strain and Pru strain,respectively,and successfully obtained RHΔCTrp26,RHΔCTrx1,PruΔCTrp26 and PruΔCTrx1 deletion strains.Immunofluorescence analysis showed that CTrp26 and CTrx1 were both located in the cytoplasm of T.gondii tachyzoites.Replication,plaque,egress,detection of ROS level and total antioxidant capacity(T-AOC),bradyzoite differentiation,and mouse virulence assays were performed on CTrp26 or CTrx1 deletion strains,and it was found that CTrp26 or CTrx1 deletion did not affect the replication,plaque formation,egress and mouse virulence of RH and Pru strains,also did not affect the ROS level and T-AOC in RH strain and did not change the ability of bradyzoite differentiation in Pru strain.2.The basic biological function of Trx2 was examined in this research.Trx2 deletion strain and complement strain in T.gondii RH strain were generated by using the CRISPR-Cas9 technology.Immunofluorescence analysis showed that Trx2 was located in the cytoplasm of T.gondii tachyzoite.The replication,plaque,T-AOC detection and mouse virulence assays of Trx2 deletion strain showed that deletion of Trx2 did not affect RH strain to replicate,form plaque and maintain T-AOC;however,Trx2 deletion can reduce the virulence of the T.gondii RH strain.The survival rate of mice inoculated with RHΔTrx2 tachyzoites was 20% at day 30 after infection,while the mice inoculated with RH wild strain tachyzoites and RHΔTrx2C tachyzoites all died within 13 days.Then the survived mice were inoculated with 500 tachyzoites of the RH wild strain.The mice immunized with RHΔTrx2 tachyzoites did not die,while the unimmunized mice all died within 9 days after inoculation with RH wild strain tachyzoites.The mouse virulence experiment showed that the virulence of RHΔTrx2 tachyzoites was lower than that of the RH wild strain.Mice immunized with the RHΔTrx2 strain developed immunity against re-infection with the RH wild strain.In summary,this study successfully constructed three Trx-deficient(CTrp26,CTrx1 and Trx2)strains using CRISPR-Cas9 technology,and examined the phenotypes of the deletion strains.The results showed that CTrp26,CTrx1 and Trx2 were all located in the cytoplasm of T.gondii tachyzoites,and the deletion of CTrp26 or CTrx1 did not affect the replication,plaque formation,egress,level of ROS and T-AOC,bradyzoite differentiation and virulence of T.gondii;Trx2 deletion did not affect the replication of T.gondii,the formation of plaques and T-AOC,but the deletion of Trx2 attenuated the virulence of the RH strain of T.gondii,thus,the RHΔTrx2 strain may be used as a candidate strain for attenuated vaccine against toxoplasmosis.