Validation of Toxoplasma Gondii Genes that Determine Fitness In Ifnγ-Activated Macrophages

Abstract

Toxoplasma gondii is an important pathogen to human and veterinary health. This study validated two T. gondii genes that were determined to confer fitness in IFNγ-stimulated macrophages from a loss-of-function CRISPR screen. Using CRISPR/Cas9, individual knockout of candidate genes was generated. Plaque assay was used to determine if knockouts had general fitness defects. IFNγ stimulation of murine and rat BMDMs in luciferase assays was used to determine the knockout phenotype in vitro. The phenotype of candidate genes was determined in vivo during acute murine infection. Finally, the genes were endogenously tagged and their localization in the parasite was determined by immunofluorescent assays. Positive clones of single-gene knockout parasites were successfully obtained and determined not to have general growth defects in HFF cells. Clone ∆TGGT1_205350 #1-6F was determined to have reduced growth with increased concentration of IFNγ in rat BMDMs, while all clones of ∆TGGT1_232670 grew unrestricted like the wild-type parasites. Murine BMDMs were consistently unresponsive to IFNγ stimulation hence growth phenotype of candidate genes could not be assessed. Murine infection revealed that TGGT1_205350 was dispensable for parasite fitness during acute infection, while the likely subcellular localization of the TGGT1_205350 was observed to be in the Golgi apparatus. In conclusion, candidate gene TGGT1_205350 was demonstrated to confer fitness in IFNγ-stimulated rat BMDMs and it is possibly localized in the Golgi compartment of T. gondii.