REGULATORY FACTORS THAT DETERMINE TOXOPLASMA BRADYZOITE TO MEROZOITES CONVERSION.

Project SummaryThe parasite Toxoplasma gondii can cause severe disease in immunocompromised patients and fetuses. Inintestinal epithelial cells of cats, its definitive host, Toxoplasma converts into merozoites, which are an asexualform that ultimately generates gametes that upon fusion will form hundreds of millions of oocysts. Theseoocysts are shed within cat feces, are highly stable in the environment, extremely resistant to inactivationprocedures, and even a single oocyst is infectious. Therefore the generation of oocysts plays a crucial role inthe prevalence of toxoplasmosis. Despite the crucial epidemiological role of the cat intestinal stages they arethe least characterized, mainly because these stages are not cultivatable in vitro and difficult to access in vivo.Our overall goal is to identify and characterize the Toxoplasma regulatory factors that drive thedevelopment of the initial cat intestinal stages. We hypothesize that by upregulating multiple Toxoplasmaregulatory factors that are normally only expressed in the cat intestine we can learn which factors play animportant role in Toxoplasma conversion into the initial merozoite stage. In our first aim we will develop 15transgenic parasite lines in each of which we can inducibly express a key Toxoplasma regulatory factor in vitro,which is normally only expressed in the cat intestine. Upon expression of these regulatory factors we willassess if they induce expression of genes that are normally only expressed in the cat intestine using high-throughput RNA sequencing. In our second aim we will test the hypothesis that the combined upregulation ofmultiple regulatory factors characterized in Aim 1 can convert Toxoplasma to merozoites. We will transientlytransfect a merozoite reporter parasite line with a pool of all the regulatory factors from Aim 1 cloned behind aconstitutive promoter. Our reporter parasites will express a drug-resistance and fluorescence marker driven bya merozoite promoter allowing us to visualize and select parasites that have successfully switched tomerozoites. We will determine the relative frequency of parasites that successfully convert into merozoites. Wewill subsequently investigate the effect of individual factors, by withdrawing them from the transfection pool, onthe formation of merozoites. This will identify what factors are essential for the stage conversion process. Wewill perform transcriptional profiling to confirm the parasite has converted to merozoites. If the complete pool ofregulatory factors together does not induce merozoite stage conversion we will add small molecules that targethistone modifying enzymes and simulate a more cat-like gut environment by infecting polarized cat epithelialcells with the transfected parasites. We will also perform a small CRISPR/CAS9 screen to identify putativerepressors of merozoite stage conversion. This high risk - high reward exploratory R21 study uses innovativeapproaches that have the potential to discover how to culture merozoites in vitro, which could eventuallycontribute to the development of novel transmission blocking drugs or vaccines against Toxoplasma.