The role of the GPI in Toxoplasma gondii pathogenesis and mRNA vaccines

A fully protective vaccine to prevent human parasitic disease has remained elusive. Our investigation of acquired immunity to Toxoplasma gondii that follow either natural infection or vaccination has led us reconsider the role of glycosylphosphatidylinositols (GPIs) in parasite pathogenesis and vaccination. GPIs are highly conserved anchors for eukaryotic cell surface proteins. T. gondii"s plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in T. gondii has remained largely unknown without an understanding of its biosynthesis. We became curious of the gene Tg_207750 as it was one of the most differentially expressed genes between type I strains that either evade immunological memory (GT1) or are controlled by it (RH) and is a predicted glycosyltransferase that adds hexoses to mannose chains. Here we report the identification and disruption of Tg_207750 ("PIGJ") and Tg_266320 ("PIGE") GPI sidechain glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. Parasite numbers are not affected by Δpigj early in the infection, suggesting a breakdown of infectious tolerance, which is dependent on host galectin-3 but not TLR2/4 GPI interacting partners. In contrast, humoral recognition of GIPL is entirely dependent on the terminal Glucose as inferred from PIGE mutants. Furthermore, we present evidence that the GPI of type I RH but not GT1 strains lacks sidechains, further implicating this pathway in secondary infection immunity. To directly investigate the role of the GPI in humoral responses, we have tested SAG1 immunization with and without GPI attachment through mRNA vaccination, which revealed a fundamental requirement for the GPI in eliciting humoral responses to GPI-anchored proteins. Ongoing results will be discussed.