Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China

Several rhoptry proteins (ROPs) have been confirmed to be critical virulence factors of Toxoplasma gondii strains from North America and Europe. The two active kinases ROP17 and ROP18, and pseudokinase ROP5 were thought to be the key determinants of parasites' virulence in laboratory mice. Given the genetic diversity of Toxoplasma strains from different geographical regions, the virulence determinants in other strains, particularly the ones that are phylogenetically distant to the North American and European strains, are yet to be elucidated. In this study, we sought to examine the contribution of three known virulence factors to the virulence of a type I strain (T.gHB1) isolated from Central China. We deleted ROP17 and ROP18 individually, as well as in combination with GRA7 by the CRISPR-Cas9 system in this local isolate. Subsequent virulence tests in mice indicated that deletion of GRA7, ROP17, or ROP18 in T.gHB1 showed similar attenuation in mice as the type I RH strain lacking the corresponding proteins. However, in contrast to the reported double knockouts in RH, double deletions of GRA7 plus ROP17 or GRA7 plus ROP18 in T.gHB1 did not show significant further virulence attenuation compared to the ROP17 or ROP18 single knockouts. These results indicated that GRA7, ROP18 and ROP17 may play different roles in virulence determination in genetically diverse strains of Toxoplasma.