[Generation of a dense granule protein 3 gene-deficient strain of and its virulence testing].

Objective: To generate a dense granule protein 3 () gene-deficient mutant of the ME49 strain and to test the virulence of the mutant.

Methods: Gene-deficient parasites were generated with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Guide RNA (gRNA) was designed using the E-CRISPR software, and mutated on the pSAG1::Cas9-U6::sgUPRT plasmid using the Q5 site-directed mutagenesis kit to generate the pSAG1::Cas9-U6::sgGRA3 plasmid. A donor plasmid containing gene upstream sequences, pyrimethamine resistant gene dihydrofolate reductase-thymidylate synthase () and gene downstream sequence was generated, and donor DNA was amplified using PCR assay. The pSAG1::Cas9-U6::sgGRA3 plasmid and donor DNA were electroporated into tachyzoites of the wild-type ME49 strain. Then, parasite suspensions were inoculated into human foreskin fibroblast (HFF) cells and screened with pyrimethamine to yield pyrimethamine-resistant parasites for monoclonal screening. The gene deficient monoclonal strain (ME49Δ) of was identified using PCR and Western blotting assays, and the expression of GRA3 protein was determined in the ME49Δ strain using Western blotting. Subsequently, 1 000 freshly lysed tachyzoites of ME49 and ME49Δ strains were transferred to 12-well plates seeded with HFF cells, and incubated at 37 °C containing 5% CO for 7 days, and the number of plaques was counted by staining with crystal violet solutions. HFF cells infected with tachyzoites of ME49 and ME49Δ strains were stained using Giemsa solutions, and the numbers of cells containing 1, 2, 4, and > 4 parasitophorous vacuoles were counted. In addition, the survival rates of C57BL/6 mice infected with ME49 and ME49Δ strains were compared 35 days post-infection.

Results: PCR assay revealed successful amplification of both the upstream and downstream homologous arm bands of the gene in the ME49Δ strain, and no corresponding bands were amplified in the ME49 strain. The band was amplified in the ME49 strain, and the band, rather than band, was amplified in the ME49Δ strain. Western blotting determined absence of GRA3 protein expression in the ME49Δ strain. Crystal violet staining showed that the ME49 strain produced more plaques than the ME49Δ strain [(352.67 ± 26.39) plaques vs. (235.00 ± 26.29) plaques; = 5.472, < 0.01], and Giemsa staining revealed that the proportion of parasitophorous vacuoles containing at least four tachyzoites was higher in HFF cells infected with the ME49 strain than in those infected with the ME49Δ strain [(75.67 ± 2.52)% vs. (59.67 ± 2.31)%; = 8.113, < 0.01], and the proportion of parasitophorous vacuoles containing at least 1 or 2 tachyzoites was higher in HFF cells infected with the ME49 strain than in those infected with the ME49Δ strain [(24.33 ± 2.52)% vs. (40.33 ± 2.31)%; = -8.113, < 0.01]. In addition, mice infected with the ME49 and ME49Δ strains started to die 8 and 9 days post-infection, and the 35-day mortality rates of mice infected with ME49 and ME49Δ strains were 10.00% and 70.00% post-infection (χ = 6.762, < 0.01).

Conclusions: The ME49Δ strain has been successfully generated, and GRA3 protein may increase the virulence of the ME49 strain.