Screening And Functional Study Of Genes Against Oxidative Damage In Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular Apicomplexa protozoan that can infect the nucleated cells of nearly all warm-blooded animals,seriously endangering human health.T.gondii infection of host leads to the initiation of innate immune response and the immunocyte is activated to creates massive amounts of reactive oxygen species(ROS).ROS has anti-Toxoplasma activity mainly by inducing oxidative stress.The antioxidant system of T.gondii plays an important role in the defense against oxidant stress imposed by the host.However,there is still a lack of comprehensive research on anti-oxidation system,and the critical ones that function in the ROS stress response are still poorly known.Here,we performed a genome-wide CRISPR/Cas9 loss-of-function screen in the T.gondii RH strain to identify potential genes contributing to the ROS stress response,and explore the biological function of new genes preliminarily.This study will provide the basis for further study on the mechanism of resistance to oxidative stress in T.gondii.To determine the optimal concentration and time of H₂O₂ on cells and T.gondii,the oxidative stress model of Vero cells was established.Vero cells were treated with different concentrations of H₂O₂(100?200?300?400?500?600?800?M)and cell viability was evaluated by CCK-8 assay.The intracellular ROS level was measured by the fluorescent probe,DCFH-DA.The optimal concentration of H₂O₂ for Vero cells was determined to be 200?M.Next,RH strain,ROP5 gene deletion strain and TR gene deletion strain were used as experimental strains to evaluate the effect of H₂O₂ on extracellular T.gondii,to determine the optimal incubation time of H₂O₂.The results showed that 30 min was the optimal treatment time to incubate T.gondii with H₂O₂.Then,the effect of H₂O₂ on the proliferation was evaluated by adding H₂O₂ to the culture medium.The results showed that 200?M H₂O₂significantly inhibited the proliferation of intracellular TR-KO strains,and had no significant effect on RH strains and ROP5-KO strains.Therefore,T.gondii were incubated with 200?M H₂O₂ for 30 min,and H₂O₂ was added in the medium as the conditions for screening antioxidant related genes.CRISPR/Cas9 genome-wide screening was used to screen and identify antioxidant genes.Cas9-expressing strain(RH/Cas9)was constructed by co-transfecting the RH strain with the p Cas9/chloramphenicol acetyltransferase and p Cas9/decoy plasmids.Next,the sg RNA library was electroporated into to the RH/Cas9 strain.The mutant pool was obtained after screening by pyrimethamine.According to the above screening conditions,200?m H₂O₂ was used to screen the antioxidant genes of T.gondii.Analyzing the enrichment and loss of sg RNA,5 hypothesis proteins(HPs)were selected for validation test.Five HP deletion strains(HPs-KO)were constructed using RH/Cas9strain.The sensitivity of HPs-KO strain to H₂O₂ was verified,and the antioxidant capacity,invasion and proliferation capacity of the mutants,and its virulence to mice were determined.The results showed that the deletion of HPs increased the sensitivity of T.gondii to H₂O₂ in different degrees.Compared with other mutants,HP1-KO strain were the most sensitive to H₂O₂.The antioxidant capacity,the invasion and proliferation of the HPs-KO strains were decreased and the virulence in mice was reduced.To further study the function of HP1,prokaryotic expression of HP1 were carries out and polyclonal antibodies were prepared.The localization analysis of HP1 protein in parasites and co-immunoprecipitation(CO-IP)were performed by HP1 mouse polyclonal antibodies.HP1 gene deletion strain(HP1-KO)was constructed by RH strain.Complementary strain(HP1-CO)was further obtained.The effect of HP1 deletion on the expression of related genes in parasites was investigated using RNA-Seq.The results showed that HP1 was localized on the cell membrane of T.gondii.The results of mass spectrometry showed that HP1 related proteins were mainly involved in ribosomes and various metabolic pathways,such as Glycolysis/Gluconeogenesis,Glutathione metabolism and the pentose phosphate pathway.RNA-Seq results showed that there were 710 differentially expressed genes,among which 48 were upregulated and 662 were downregulated in the HP1-KO strain compared to wild-type RH strain.Gene Ontology(GO)analysis indicated that differentially expressed genes were enriched in‘metabolic process',‘cellular processes',‘binding'and‘catalytic activity'.The results of KEGG pathway analysis showed differentially expressed genes involved in the JAK-STAT signaling pathway,the phosphatidylinositol signaling system,cell senescence and fatty acid biosynthesis.The above results indicated that HP1 mainly affected the metabolic process of T.gondii,which was different from the ability of CAT to decompose H₂O₂.Next,a double gene deletion of HP1 and CAT(HP1/CAT-KO)was constructed using HP1-KO strain.Next,the sensitivity of HP1/CAT-KO to H₂O₂,the proliferation of HP1/CAT-KO in mice and the virulence of HP1/CAT-KO to mice were detected.The results showed that HP1/CAT-KO strain was successfully constructed and the sensitivity of HP1/CAT-KO to H₂O₂ was significantly higher than that of single deletion strain.The proliferation rate of HP1/CAT-KO in mice was significantly lower than that of RH strain and single deletion strain.The survival time of mice infected with HP1/CAT-KO strain was prolonged.Finally,a preliminary functional study was conducted on the AKHP gene found in genome-wide CRISPR/Cas9 screening.Bioinformatics analysis showed that AKHP protein has a typical 2-Cys-Prx domain.The location analysis showed that AKHP was mainly localized in the cytoplasm of T.gondii.AKHP gene deletion(AKHP-KO)and complementary strains(AKHP-CO)were constructed by CRISPR/Cas9 system.The invasion and proliferation ability,sensitivity to H₂O₂ and antioxidant ability of different strains were determined.The m RNA expression of other antioxidant genes in AKHP-KO was determined by q RT-PCR.The results showed that AKHP was AKHP-KO strain and AKHP-CO were successfully constructed.It was found that deletion of AKHP significantly reduced the proliferation ability of T.gondii,significantly increased the sensitivity to H₂O₂,and decreased the antioxidant capacity of the parasite.Compared with the RH strain,the expression of Prx2 in the AKHP-KO strain was significantly increased.In summary,this study successfully screened and identified 5 unknown antioxidant related genes and AKHP gene possessed typical 2-Cys-Prx domain.The functional of HP1 and AKHP genes were preliminary studied,providing a basis for further studies on the mechanism of resistance to oxidative stress in T.gondii.