N-Glycosylation in Piroplasmids: Diversity within Simplicity

Abstract

N-glycosylation has remained mostly unexplored in Piroplasmida, an order of tick-transmitted pathogens of veterinary and medical relevance. Analysis of 11 piroplasmid genomes revealed three distinct scenarios regarding N-glycosylation: Babesia sensu stricto (s.s.) species add one or two N-acetylglucosamine (NAcGlc) molecules to proteins; Theileria equi and Cytauxzoon felis add (NAcGlc)₂-mannose, while B. microti and Theileria s.s. synthesize dolichol-P-P-NAcGlc and dolichol-P-P-(NAcGlc)₂ without subsequent transfer to proteins. All piroplasmids possess the gene complement needed for the synthesis of the N-glycosylation substrates, dolichol-P and sugar nucleotides. The oligosaccharyl transferase of Babesia species, T. equi and C. felis, is predicted to be composed of only two subunits, STT3 and Ost1. Occurrence of short N-glycans in B. bovis merozoites was experimentally demonstrated by fluorescence microscopy using a NAcGlc-specific lectin. In vitro growth of B. bovis was significantly impaired by tunicamycin, an inhibitor of N-glycosylation, indicating a relevant role for N-glycosylation in this pathogen. Finally, genes coding for N-glycosylation enzymes and substrate biosynthesis are transcribed in B. bovis blood and tick stages, suggesting that this pathway is biologically relevant throughout the parasite life cycle. Elucidation of the role/s exerted by N-glycans will increase our understanding of these successful parasites, for which improved control measures are needed.

Keywords: Babesia; Cytauxzoon; N-glycan; Theileria; dolichol; piroplasmids; sugar nucleotides.

Figure 1

N-glycosylation pathways and end products in piroplasmids. (A) Predicted pathway for the N-glycosylation of an exported protein in Theileria equi and Cytauxzoon felis: (1) Synthesis of dol-P-NAcGlc by Alg7; (2) synthesis of dol-P-P-(NAcGlc)₂ by Alg13/Alg14 (both processes occur at the cytoplasmic leaflet of the rough endoplasmic reticulum (RER) membrane and utilize UDP-NAcGlc as sugar donor); (3) synthesis of dol-P-P-(NAcGlc)₂-Man by Alg1; (4) flipping to the luminal leaflet of the RER by a flippase; (5) transfer of (NAcGlc)₂ to a signal peptide-bearing nascent protein by oligosaccharyl transferase activity (OST); (6) cleavage of signal peptide liberating the recently synthesized N-glycosylated protein (green); (7) dephosphorylation of dol-P-P to dol-P; (8) flipping of dol-P to the cytoplasmic leaflet of the RER for reuse. Babesia s.s. lack step 3, while Theileria s.s. and B. microti lack steps 3 and 5. SP: Signal peptide. (B) End products resulting from three different N-glycosylation enzyme sets: dol-P-P-(NAcGlc)₂, and (NAcGlc)₂ or Man-(NAcGlc)₂ modifying a protein. (C) graphical references.

Figure 2

Detection of short NAcGlc glycans in B. bovis mz. Fixed smears of B. bovis-infected erythrocytes were incubated with biotin-labeled GSLII lectin, followed by FITC-streptavidin and 4,6-diamidino-2-phenylindole (DAPI) for nuclei Scheme 1000× in an epifluorescence microscope using filters to detect FITC (a) or DAPI (b). (c) Merged image.

Figure 3

Comparison of the transcription levels of N-glycosylation -related genes in blood and tick B. bovis stages. (A) N-glycosylation pathway-encoding genes. (B) Genes related to the biosynthesis of UDP-NAcGlc (S) and dol (D), the substrates for N-glycan synthesis in B. bovis. (C) Genes related to the synthesis of GDP-Man, which in B. bovis is only used as sugar donor for GPI biosynthesis. Red bars: Blood stages; blue bars: Tick stages.

Figure 4

Dose-dependent effect of Tun on the in vitro growth of B. bovis merozoites. Percentages of parasitized erythrocytes (PPE) were calculated by microscopic observation of Giemsa-stained smears withdrawn at T0 (light blue bars) and after 72 h (dark green bars) of cultivation. Bars show the average + SD of three independent cultures. Asterisks mark significant decreases with respect to the untreated cultures (0 μM): * p < 0.05; ** p < 0.001.