Functional Analysis Of The ANK1 Gene During Toxoplasma Growth And Development

Toxoplasma gondii is an important zoonotic protozoan with a wide host range, it can infect all warm-blooded animals and humans. Appoximately one quarter of the world population are infected with toxoplasma. In immunocompetent hosts, acute toxoplasma infection is quickly shifted to chronic infection due to the immune function of the hosts. Under such immune pressure, the parasites are converted from the rapidly replicating tachyzoites to slowly growing bradyzoites, which exist in the form of tissue cysts and cause lifelong chronic infection in hosts. However,when the host immune system is compromised, tissue cysts can reactivate and convert from bradyzoites to tachyzoites, leading to acute infection. The conversion between tachyzoites and bradyzoties plays an important role in toxoplasma transmission and pathogenesis. There are about 400 genes,whose expression levels show significant changes during this conversion process, but their functions are largely unknown. Functional dissection of these genes can offer significant insights into the mechanisms governing bradyzoite development and provide theoretical basis for effective T.gondii vaccine design.ANK1 is a tetratricopeptide repeat(TPR) structural motif containing protein that is dramatically up-regulated during bradyzoite formation. To test the function of ANK1, we knocked out this gene in the PRU strain using the CRISPR/Cas9-based system. Subsequent plaque assay,replication assay and virulence test indicated that the ANK1 mutant has defects in both tachyzoite growth and bradyzoite development.(1) Prokaryotic expression of full-length and N-terminal fragment of ANK1.Recombinant plasmids pE-SUMO-ANK1-Nter and pE-SUMO-ANK1 expressing N-terminus and full-length ANK1 respectively were successfully constructed. The two plasmids were individually transformed into BL21(DE3)competent cells and the cultures were induced with IPTG at 37℃ for recombinant protein expression. Under such conditions, both ANK1 N-terminal fragment and full-length protein can be expressed effectively, but the ANK1 full-length protein was mainly expressed in inclusion bodies. To optimize the expression conditions to increase the solubility, we grew the cultures to OD600 = 1.5 and then induced with final concentration of 0.1mM IPTG at 16℃. With this condition, most recombinant proteins were soluble in supernatant.(2)Construction of the ANK1 knockout strainANK1 was disrupted using CRISPR/Cas9-based system. First, the ANK1 targeting CRISPR plasmid pSAG1-Cas9-U6-gANK1 and the homology template plasmid pANK1-DHFR plasmid were constructed. Subsequently the CRISPR plasmid and the homology template were co-transfected into the PRU strain.The parasites were then selected with pyrimethamine and single clones were obtained in 96-well plate by limiting dilution. Single clones were screened by diagnostic PCRs and the results indicated that ANK1 has been successfully disrupted in PRU.(3) Phenotypic analysis of ANK1 mutantsPlaque assay was used to compare the difference in parasites growth, ANK1 mutant growed significantly more slowly than the parental line. Replication results indicated that ANK1 mutant displayed obvious intracellular replication defects. Purified tachyzoites from WT and mutant strains were intraperitoneally injected into ICR mice and their survival was monitored for 30 days. Results indicated that the virulence of ANK1 knockout in ICR mice was decreased significantly. Immunization of mice with ANK1 knockout parasites offered protection to lethal doses of WT T.gondii parasites.(4)Identification of differentially expressed genes between ANK1 mutant and wild-type parasites by RNA-seq.Culture ANK1 mutant and wild-type strain in vitro to obtain tachyzoites and induction of bradyzoites development was also conducted in these two strains. Extract the total RNA from tachzyzoites and bradyzoites respectively and determine their gene expression patterns by RNA-seq. The results indicated that: there were 430 differentially expressed genes at the bradyzoite stage between WT and ANK1 mutant, whereas 510 genes displayed expression differences at the tachyzoite stage.In conclusion, the ANK1 gene has been successfully disrupted in the PRU strain using the CRISPR/Cas9 system. Compared with the WT PRU strain, ANK1 mutant showed defects in growth, replication and virulence. ANK1 mutant may also be used as a live attenuated vaccine. RNA-seq analysis identified differentially expressed genes between WT and ANK1 mutant at both tachyzoite and bradyzoite stages, which provided insights to further dissect the working mechanisms of ANK1 in regulating parasite growth and development.