Effect of deletion of gra17 and gra23 genes on the growth, virulence, and immunogenicity of type II Toxoplasma gondii

The protozoan parasiteToxoplasma gondiisecretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to deletegra17orgra23gene inT. gondiiPru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Delta gra17and Pru:Delta gra23mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion ofgra17abrogated acute infection and blocked cyst formation. Although the deletion ofgra23caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Delta gra17induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-gamma]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain ofT. gondii. These findings show that GRA17 and GRA23 play important roles inT. gondiichronic infection and that irreversible deletion ofgra17inT. gondiitype II Pru strain can be a viable option for stimulating protective immunity toT. gondiiinfection.