Characterization of strain-specific phenotypes associated with knockout of dense granule protein 9 in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of mammals and cause toxoplasmosis. Dense granule proteins play major structural functions within the parasitophorous vacuole (PV) and the cyst wall of T. gondii. Moreover, their particular location within the PV allows them to be involved in various interactions between parasites and the host cells. Dense granule protein 9 (GRA9) gene has been identified in T. gondii, although its role in the lytic cycle remains unclear. In the current study, the function of GRA9 in type I and type II Toxoplasma parasites was characterized. T. gondii GRA9 sequence and its expression were analyzed and derivatives of T. gondii RH and PLK strains with a null mutation in GRA9 were generated using CRISPR/Cas9 system. The phenotypes of GRA9 in wild types, knockout and complemented strains were analyzed in vitro and in vivo using Vero cells and BALB/c mice, respectively. Alignment of the amino acid sequence indicated that RH strain GRA9 contained one amino acid substitution when compared with PLK strain. Western blot analysis revealed that PLK strain had a higher expression level of GRA9 than RH strain. The phenotype analysis revealed that knockout of GRA9 in PLK parasites inhibited the plaque formation and egress from PV. Both the plaque formation and egress ability of PLK Delta GRA9 strain were restored by complementation with a synonymous allele of PLK strain GRA9. Mouse experiments demonstrated that loss of GRA9 in PLK strain significantly reduced the pathogenicity of T. gondii. However, there was no phenotypic diferences between RH and RHAGRA9 strains except the defect in host cell invasion. Overall, T. gondii GRA9 knockout only influenced the growth and virulence of PLK strain. These results indicate that GRA9 may be involved in parasite egress and virulence in mice in a strain-specific manner.