Figure 1. Toxoplasma tachyzoites metabolize unnatural sugars.

(A) Structures of peracetylated N-acetylglucosamine (Ac₄GlcNAc) and the corresponding alkynyl (Ac₄GlcNAlk) and azido (Ac₄GlcNAz) analogs used in metabolic labeling studies. The acetyl groups facilitate sugar uptake into cells and are removed by the action of non-specific esterases in the cytoplasm. An azide-functionalized acyl chain (Az-CO₂Et) used in various control experiments is also pictured. The azide and alkyne functional groups can be detected using bioorthogonal “click” chemistries. (B) Toxoplasma tachyzoites incorporate Ac₄GlcNAz in a dose-dependent manner. Parasites were incubated with unnatural sugar (0.1–5 mM) or the control sugar Ac₄GlcNAc for 8 hours, lysed, and reacted with a biotin-alk tag via “click” chemistry. The labeled proteins were separated via gel electrophoresis and detected via immunoblot with streptavidin. Equivalent protein loading was confirmed via staining with Ponceau S (Figure S1) and normalized intensities are listed. (C) Toxoplasma tachyzoites incorporate Ac₄GlcNAz in a time-dependent manner. Parasites were incubated with Ac₄GlcNAz (1 mM) or the control sugar Ac₄GlcNAc (1 mM) for 2–12 h hours, lysed, and reacted with a rhodamine-alkyne probe. Labeled proteins were separated via gel electrophoresis and analyzed via in-gel fluorescence. Equivalent protein loading was confirmed via staining with Ponceau S (Figure S4) and normalized intensities are listed. (D) Ac₄GlcNAz treatment reveals a unique glycoprotein fingerprint. Parasites were incubated with Ac₄GlcNAz (1 mM), Ac₄GlcNAc (1 h) or a non-sugar probe (Az-CO₂Et) for 8 hours, lysed, and reacted with a biotin-alk tag. Labeled proteins were analyzed via immunoblot as in (C). Equivalent protein loading was confirmed via staining with Ponceau S (Figure S7).