Construction And Biological Characterization Of Toxoplasma Gondii Dense Granule Proteins Deletion Strains

Toxoplasma gondii is an apicomplexan protozoon with a vast range of hosts,which can cause significant ecomonomic lossess to the livestock industry and considerable public health problems.Dense granule,a unique secretory organ of apicomplexan protozoa,secrets a number of dense granule proteins that play important roles in regulating signaling pathways,participating in parasite growth,maintaining PV and PVM structures.Firstly,CRISPR/Cas9-mediated non-homologous end joining technique was used to knockout the17 GRA genes(GRA11,GRA12 bis,GRA13,GRA14,GRA20,GRA21,GRA28-31,GRA33-38,and GRA40)of T.gondii RH strain(type ?).Basic biological functions of 17 ?GRA mutants were evaluated using plaque and egress assays in vitro,and virulence assay in mice.This study concluded that the deletion of these 17 GRA genes had no significant effect on the growth of T.gondii in vitro and virulence in mice.Then,RH?GRA12,Pru?GRA12 and GRA12 complement strains(RH?GRA12C or Pru?GRA12C)were constructed using the CRISPR/Cas9-mediated homologous recombination technique to explore phenotypic traits,such as plaque,replication and virulence.Results showed that there were no significant differences between knockout strains and their wild strains in proliferation and plaque formation in vitro.However,in virulence assay,the ?GRA12 parasites were significantly attenuated,and the number of brain cysts in mice had significantly reduced.These findings indicate that GRA12 is a virulence gene in type ? RH and ? Pru strains.Previous studies have shown that ROP18 is an important virulence factor in T.gondii.Hence,T.gondii RH?ROP18?GRA12 knockout strain was constructed by CRISPR/Cas9 system,which was verified by PCR amplification and western blot assay.To illustrate its protection efficacy as an attenuated vaccine,C57BL/6 mice were injected with RH?ROP18?GRA12 double deletion strain,each mouse was injected intraperitoneally with 100 tachyzoites of RH?ROP18?GRA12 or wild-type RH strain.All mice infected with 100 tachyzoites of the RH strain died within 7 days after infection.In contrast,all mice that infected with RH?ROP18?GRA12 survived.Forty-five days post infection,all survived mice were re-challenged with 100 tachyzoites of the wild-type RH strain and the survival time were observed.All mice infected with RH?ROP18?GRA12 strain survived after re-infected with 100 RH tachyzoites,however the na?ve mice that infected with 100 RH died within 7 days after infection.These results showed that the double deletion of ROP18 and GRA12 reduced the virulence significantly,and immunization with RH?ROP18?GRA12 tachyzoites induced strong immunity to protect mice from infection with RH strains,suggesting that ?ROP18?GRA12 mutant is a promising attenuated vaccine candidate strain.In conclusion,18 GRA knockout strains were successfully constructed using CRISPR/Cas9 technique,and their basic biological functions were elucidated.We found that the knockout of 17 GRA(GRA11,GRA12 bis,GRA13,GRA14,GRA20,GRA21,GRA28-31,GRA33-38,and GRA40)genes have no significantly effect on Toxoplasma growth in vitro and virulence in mice,but GRA12 is avirulence gene in type I RH and type ? Pru strains of T.gondii.RH?ROP18?GRA12 strain was contructed and found to be a promising attenuated vaccine candidate strain.These results laid a foundation for future research on anti-T.gondii vaccine.