Loss of rhoptry protein 9 impeded Toxoplasma gondii infectivity

Toxoplasma gondii is an obligatory intracellular parasite that critically depends on active invasion and egress from infected host cells to complete its propagation cycle. T. gondii rhoptry proteins (TgROPs) are virulent factors associated with host cell invasion, growth. In this study, we analyzed the functions of ROP9 in the process of T. gondii infection. The TgROP9 knockout RH strain (RH Delta ROP9) and its recovery strain (RH-ReROP9) were constructed using the CRISPR/Cas9 system. The invasion, proliferation, and egress efficiency of the RH Delta ROP9 strain were evaluated and their pathogenicity to mice was analyzed. Compared with RH wild-type (RH-WT) and RH-ReROP9 strains, the invasion percentage of RH Delta ROP9 to Vero cells was reduced by about 28.0% (p< 0.01) at 1.5 h, and the relative proliferation percentage was decreased by about 35.0% (p < 0.01) after infection with 10(2) or 10(3) parasites. In addition, the RH Delta ROP9 strain also showed prolonged egress time from host cells. The survival time of the mice (12.6 +/- 1.6 or 10.1 +/- 1.1 days) were delayed (p < 0.001) after infection with either 200 or 1000 RH Delta ROP9 parasites. These evidences suggested that ROP9 facilitated T. gondii infection in vitro and in vivo.