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