Biological Function Of Adenylosuccinate Lyase And Adenosine Kinase During Toxoplasma Gondii Growth And Development

Toxoplasma gondii is a protozoan parasite,occurring worldwide,endangers human health and causes enormous economic losses to the Agriculture.Toxoplasma gondii has two forms of tachyzoites and bradyzoites.The rapid reproduction of tachyzoites in the intermediate host will cause acute infection of Toxoplasma gondii.In severe cases,it will manifest as toxoplasmosis,encephalopathy and even abortion.In the immunocompetent host,Toxoplasma gondii can differentiate to form bradyzoite become tissue cysts cause long-term chronic infection of the host.Studies on the metabolism of bradyzoites and tachyzoites help design of drug targets for Toxoplasma gondii and development of vaccines.Nucleotide synthesis is essential for the survival of T.gondii,the majority of nucleic acid metabolism-related enzyme is expressed in Toxoplasma gondii bradyzoite and tachyzoite stage.Hypoxanthine xanthine guanine phosphoribosyltransferase(HGGPRT)involved in the synthesis of Toxoplasma purine nucleotides is widely used as a drug target because of its redundancy with adenylate kinase(AK).In addition,many enzymes are involved in the metabolism of Toxoplasma purine nucleotides,and most of the functions are unclear.ADSL and AK are involved in the synthesis of adenine nucleotides of Toxoplasma gondii.In this study,we selected ADSL and AK gene for biological function research to analyze the molecular mechanism of Toxoplasma gondii nucleicacid precursor material synthesis.Perform genetic operations and functional research on ADSL and AK.Specific work includes the following aspects:(1)Construction and functional study of ADSL and AK gene knockout strainsSingle-deletion and double-deletion strains of ADSL and AK were successfully obtained using CRISPR/CAS9 gene editing technology,and related phenotypic experiments were performed.Through replication and plaque experiments,it was proved that the growth rate of Toxoplasma gondii was slowed down after single knockout of ADSL and AK,and the growth defect of double-deleted strains was more obvious.The virulence experiments in mice proved that the virulence of Toxoplasma gondii was weakened after single knockout of ADSL.However,increasing the infective dose ofΔADSL strain to ICR mice found no significant difference in virulence and wild type.Under the same conditions,the infection dose ofΔADSLΔAK strain was increased to 10^~5.The infected mice had no obvious clinical symptoms and the survival rate was100%.Reproduction of parasites in vivo experimental results show that compared to the wild-typeΔADSL and wild strains,ΔADSLΔAK strains has a reproduction defect in the vivo,which may be the reason for its weakened virulence and reduced cysts.In summary,the absence of ADSL and AK have certain effect on the in vitro and in vivo reproduction of Toxoplasma gondii.(2)Adenylate succinate lyase(ADSL)deficient strains provide good immune protection as live attenuated vaccines100 Me49ΔADSL tachyzoites infected mice weakened Toxoplasma gondii virulence and reduced cyst formation.In this study we evaluated the immunoprotective properties of Me49ΔADSL.Mice immunized withΔADSL can resist the re-infection of Toxoplasma gondii I(RH),II(ME49),III(VEG)wild-type strains,and provide100%immune protection in the short and long term of infection.High levels of specific Ig G antibodies can be detected in the serum of mice immunized withΔADSL.Further research found that sera from mice immunized with Me49ΔADSL were collected at 30d and 70d for cytokine detection,and the results proved that the immunized host produced high levels of cytokines INF-γand IL-12.The spleen cells of mice immunized for 150 days were cultured in vitro and stimulated with the soluble antigen of Toxoplasma gondii to produce high levels of INF-γand IL-12.The IL-12-INF-γaxis plays a key role in controlling the infection of Toxoplasma gondii.In conclusion,in evaluating the immune protection of Me49ΔADSL strains in mice,cellular immunity may play a dominant role.To sum up,the study of ADSL and AK in the process of purine nucleotide synthesis found that it is very critical for the growth of Toxoplasma gondii in vitro and in vivo.The mutants lacking ADSL are good candidates for live vaccines.