The only commercial vaccine against Toxoplasma gondii is the attenuated S48 strain (Toxovax®) used to protect pregnant ewes from congenital toxoplasmosis. Our research aims to understand the genotypic and phenotypic mechanisms governing S48's attenuation, with potential implications for identifying novel genes crucial to parasite persistence and uncovering new therapeutic targets. Furthermore, understanding the genetic basis of T. gondii attenuation can inform the development of Toxoplasma as a protein delivery vehicle, in both humans and animals.

We show S48 has an incomplete differentiation phenotype in vitro, where it is not able to fully differentiate into the persistent bradyzoite form. This informed our search for identifying genetic factors responsible for its attenuation. We assembled a high-quality genome of the S48 strain using Oxford Nanopore long reads and DNBseq short reads. SNP calling revealed over 500,000 total mutations, from which we identified nine key predicted loss-of-gene-function mutations unique to S48 when compared to five other normally differentiating Toxoplasma strains.

From these nine genes, we selected three genes with unknown functions to knock out and tag in the cystogenic ME49 strain. We aim to investigate the roles these genes play in bradyzoite differentiation and elucidate the functions of these previously uncharacterized genes. Among the nine genes, two have been shown to have a significant involvement in bradyzoite differentiation. We plan to use Cosmids to restore the function of these two genes in S48 and evaluate their impact on both bradyzoite differentiation and viability. The initial characterization of all genetically modified strains will primarily focus on discerning the effects of gene knockouts on parasite differentiation, including cyst formation and the expression of bradyzoite genes.

Simultaneously, we aim to use RNA sequencing to understand the stage conversion progression from tachyzoites to bradyzoites in S48, compared to ME49.