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.
Highlights
Toxoplasma gondii is an obligate intracellular protist classified as a Category B pathogen on NIAID’s list of emerging infectious diseases [1]
Using GDP-Glo and UDP-Glo assays, in which hydrolysis of the nucleotide sugar produces a luminescent signal, we showed that His6TgSPY (Fig. S1), in the absence of an acceptor substrate, hydrolyzed GDP-Fuc but not UDPGlcNAc, GDP-Man, or UDP-Gal (Fig. 1A) [32]
To detect the presence of fucitol, the product mixture from an autofucosylation reaction conducted in the presence of GDP-[3H]Fuc was subjected to β-elimination, and the released material was chromatographically analyzed by High-Performance Anion Exchange Chromatography (HPAEC)
Summary
The OFT activity of His-tagged TgSPY (His6TgSPY) purified from the cytosol of bacteria was shown in four ways. While there was no increase in the number of AAL-labeled assemblies in the complemented SPY knockout strain (Δspy::TgSPY-MYC3) versus wild-type, there was an increase in the overall intensity of the AAL labeling (Fig. 2E). Based on clonal plaque formation in fibroblast monolayers (Fig. 2H), there was a modest decrease in growth in Δspy versus wild-type (RH) tachyzoites, confirming a previous report [31], that was rescued in the complemented Δspy::TgSPY-MYC3 strain. These complementation studies confirm that TgSPY is the OFT that modifies nucleocytosolic proteins recognized by AAL and provide additional evidence that AAL-labeled assemblies colocalize with NPCs [22]. The point mutations E692K and G695D correspond to mutations in 2 A. thaliana spy hypomorphic alleles, spy (G570D) and spy (E567K), that have each been shown to result in
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