The nucleocytosolicO-fucosyltransferaseSpindlyaffects protein expression and virulence inToxoplasma gondii

Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animalsO-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogenToxoplasma gondiitransfers a different sugar, fucose, to proteins involved in transcription, mRNA processing and signaling. Knockout experiments showed thatTgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclearO-fucosylation. Here we verify thatTgSPY is the nucleocytoplasmicO-fucosyltransferase (OFT) by 1) complementation withTgSPY-MYC₃, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability toO-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified byO-Fuc are important for tachyzoite fitness,O-fucosylation byTgSPY is not essential. Growth of Δspytachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified withO-fucose. IntactTgSPY-MYC₃localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspytachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitnessin vitroand in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization ofO-fucosylated proteins, suggest thatTgSPY controls the levels of proteins to be held in reserve for response to novel stresses.