Sugar-coated parasites: Fucosylation in snail-associated larvae of the human blood fluke Schistosoma mansoni

Fucosylated carbohydrate epitopes (glycotopes) of the parasitic flatworm Schistosoma mansoni are key determinants in its development and immunobiology. Importantly, studies indicate that the expression of glycotopes---especially fucosyl glycotopes---is developmentally and gender-specifically regulated. While the schistosome glycome is perhaps the best characterized amongst invertebrates, relatively little was known until now regarding glycotope expression in the snail-associated developmental stages, namely miracidia and primary sporocysts. Moreover, the mechanisms of differential expression had not yet been examined. To begin to fill these information gaps, this research sought to 1) characterize the expression of fucosylated glycotopes in miracidia and primary sporocysts of S. mansoni and 2) identify and functionally characterize the enzymatic machinery that contributes to their production, specifically the enzymes involved in fucoconjugation, GDP-L-fucose synthesis, and GDP-L-fucose transport. Previously defined glycotope-specific antibodies were used in fluorescence microscopy and immunoblot analyses that demonstrated the regulated expression of several immunologically important glycotopes, including GalNAcbeta1-4GlcNAc (LDN), GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDN-F), Fucalpha1-3GalNAcbeta1-4GlcNAc (F-LDN), Fucalpha1-3GalNAcbeta1-4(Fucalpha1-3)GlcNAc (F-LDN-F), GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF), Fucalpha1-2Fucalpha1-3GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (DF-LDN-DF) and the truncated trimannosyl N-glycan Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn, in miracidia and primary sporocysts of S. mansoni. Additionally, a genome-wide homology-based bioinformatics approach identified multiple novel schistosome fucosyltransferases (FucTs), including six alpha3-FucTs and six alpha6-FucTs, which transfer L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor in an alpha3 or alpha6 linkage, respectively. Notably, no alpha2-FucTs were identified despite alpha2-linked fucose being abundantly expressed amongst schistosome fucoconjugates. Homology searches also identified GDP-D-mannose-4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER) and GDP-L-fucose transporter (GFT), which constitute a complete pathway for de novo GDP-L-fucose synthesis and Golgi import. Quantitative PCR analyses indicate that several fucosylation-associated genes are differentially expressed during the miracidium-to-primary sporocyst transformation, suggesting one possible mechanism for differential glycotope expression in intramolluscan schistosomes. Attempts were made to silence gene expression during larval transformation, but despite successful alpha3-FucT, GMD, GMER, and GFT knockdowns, no phenotypic changes were observed. This work provided fundamental information about a group of immunologically important schistosome genes, establishing a framework to inform and motivate future investigations that will further explore the role of fucosylation in schistosome development and immunobiology.