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 TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 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 TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy 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 TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- AAL
- Aleuria aurantia lectin
- AtSPY
- Arabidopsis thaliana SPINDLY
- CAZy
- Carbohydrate Active EnZyme
- DBA
- Dolichos biflorus agglutinin
- SPINDLY; FG-Nup
- Phe/Gly-repeats nucleoporin
- GT41
- glycosyltransferase 41
- GST
- glutathione-S-transferase
- HFF
- human foreskin fibroblast
- IDR
- intrinsically disorder region
- Me-αFuc
- α-methyl fucopyranoside
- NLS
- nuclear localization signal
- NPC
- nuclear pore complex
- O-Fuc
- O-linked fucose
- OFT
- O-fucosyltransferase
- OGT
- O-GlcNAc transferase
- SIM
- structured illumination microscopy
- SRD
- serine-rich domain
- TgGMD
- Toxoplasma gondii GDP-mannose 4,6-dehydratase
- TgGPN
- Toxoplasma gondii GPN-loop GTPase
- TgGPNΔSRD
- TgGPN missing the SRD at its N-terminus
- TgSPY
- Toxoplasma gondii SPINDLY
- TgSPY-3TPRs
- TgSPY with three C-terminal TPRs
- TPR
- tetratricopeptide repeat
- YFP
- yellow fluorescent protein