Forward genetic screen identifies SRS15 and IFT88 as critical determinants controlling Toxoplasma oocyst shedding in cats

Transmission of Toxoplasma gondii in nature is highly dependent on the parasite"s sexual cycle, which occurs exclusively in felids. Infected cats shed high numbers of infectious oocysts, which are capable of causing toxoplasmosis outbreaks in both animals and people. The molecular pathways promoting sexual stage development in T. gondii are largely unknown. To identify essential genes that regulate oocyst formation we undertook a forward genetic signature-tag mutagenesis screen, using CRISPR/Cas9 to generate a library of T. gondii strains that each possess a unique barcode and are each deficient in a single gene predicted to be highly expressed in the parasite"s sexual stages. Specifically, we used previous RNA-Seq datasets to identify 192 sexual stage-specific transcripts to test for their role promoting sexual competency. These included surface antigen genes related to 6-CYS proteins in Plasmodium, genes required to form flagella, a family of transcription factors collectively referred to as ApiAP2s, and a large family of secreted proteins expressed exclusively in merozoites (Families A-D). To identify which genes impacted oocyst formation, we pooled the 192 T. gondii knock-out (KO) strains, infected mice, then challenged cats with brain cysts to perform an input/output screen that identified sexual stage-specific genes critical for oocyst formation. Mouse brains were harvested 50 d.p.i. and split into an input sample and a cat inoculation sample. Mi-Seq analysis identified 2 genes, referred to as IFT88-, SRS15-KO that had reduced, or failed to produce oocysts in cats, respectively. When assayed individually through cats, IFT88- and SRS15-KO strains did not produce oocysts. Complementation of the IFT88 gene restored oocyst production. Cats vaccinated with either IFT88- or SRS15-KO strains were immune to homologous rechallenge, heterologous rechallenge experiments are ongoing and results will be presented. Our data demonstrate the potential efficacy of an attenuated oral vaccine to block oocyst shedding and reduce the transmission of Toxoplasma in food and water sources destined for human and animal consumption.