Abstract Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals O -GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing and signaling. Knockout experiments showed that Tg SPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O -fucosylation. Here we verify that Tg SPY is the nucleocytoplasmic O -fucosyltransferase (OFT) by 1) complementation with Tg SPY-MYC 3 , 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O -fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O -Fuc are important for tachyzoite fitness, O -fucosylation by Tg SPY is not essential. Growth of Δ spy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified with O -fucose. Intact Tg SPY-MYC 3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δ spy tachyzoites 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 fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O -fucosylated proteins, suggest that Tg SPY controls the levels of proteins to be held in reserve for response to novel stresses.

The nucleocytosolic O-fucosyltransferase Spindly affects protein expression and virulence in Toxoplasma gondii