Enabling tools forToxoplasmaglycobiology

Abstract

ABSTRACTInfection by the protozoan parasiteToxoplasma gondiiis a major health risk owing to its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Like nearly all eukaryotes,Toxoplasmaglycosylates many of its proteins and lipids and assembles polysaccharides. Unlike most eukaryotes,Toxoplasmadivides and differentiates in vacuoles within host cells. While their glycans resemble canonical models, they exhibit species-specific variations that have inhibited deeper investigations into their roles in parasite biology and virulence. The genome ofToxoplasmaencodes a suite of likely glycogenes expected to assemble a range ofN-glycans,O-glycans, aC-glycan, GPI-anchors, and polysaccharides, along with their requisite precursors and membrane transporters. To facilitate genetic approaches to investigate the roles of specific glycans, we mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute. We adapted a double-CRISPR/Cas9 strategy and a mass spectrometry-based glycomics workflow to test these relationships, and conducted infection studies in fibroblast monolayers to probe cellular functions. Through the validated disruption of 17 glycogenes, we also discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, GalNAc2- and Glc-Fuc-type O-glycans, and a nuclear O-Fuc type glycan. We describe the guide sequences, disruption constructs, and mutant strains, which are freely available to practitioners for application in the context of the relational map to investigate the roles of glycans in their favorite biological processes.