Abstract 1046 Identification of the GPI Anchor Sidechain Modifying Enzymes in Toxoplasma gondii and Their Role in Virulence

Toxoplasma gondii (T. gondii) is a protozoan parasite that is known to infect all warm-blooded animals including humans, leading to the disease known as toxoplasmosis. This pathogen has global prevalence and is known to infect 30-50% of the global human population, with more severe infection cases in South America. For the general populations containing immunocompetent individuals, Toxoplasma gondii is known to be mainly asymptomatic. However, for immunocompromised individuals, including people with chronic diseases like HIV or cancer, toxoplasmosis can lead to various medical conditions such as lung and brain damage, as well as visual impairment There are no vaccines available to prevent this infection. For that reason, our work focuses on these parasites, specifically looking at the Glycosylphosphatidylinositol (GPI)-anchor, a highly conserved glycolipid that anchors proteins to the external membrane of T. gondii via a phosphatidylinositol core and a sidechain typically composed of sugars and a phosphoethanolamine linkage. The GPI anchor is expressed in all eukaryotic cells and plays an important role in the recognition of pathogens by the immune system. The core structure is conserved across eukaryotes; however, species differ in their sidechains. In T. gondii, the GPI anchor is highly expressed. In both humans and mice, the sidechain of the T. gondii GPI is immunogenic, eliciting a robust immune response. However, the role of the glycosyl side chain of the GPI is still unknown. Our work has discovered both T. gondii sidechain modifying enzymes of the GPI; the glycosyltransferase responsible for adding the GalNAc addition to the mannose backbone of the GPI, as well as the glycosyltransferase responsible for adding Glucose to the GalNac. Using C57BL6 mice infected with knockout T. gondii CEPGFP strains for the side chain (KO for the GalNac adding glycosyltransferase: CEPGFPΔ207750 and KO for the Glucose addition glycosyltransferase: CEPGFPΔ266320) for 30 days, we found mice were susceptible to CEPGFPΔ207750. The wildtype CEPGFP strain is known to be avirulent, so this shows us that the sidechain removing T. gondii mutant has increased virulence and further experimentation shows us these mice contain increased parasite burden in the brain. Collectively, these results show that the sidechain removal led to increased virulence and illustrates the importance of the GPI sidechain modifications in the immune response and addresses future directions in treatment development. RO1 Grant.