High molecular mass glycans are major structural elements associated with the laminated layer of in vitro cultivated Echinococcus multilocularis metacestodes

Abstract

The laminated layer of the larval stage (metacestode) of the cestode parasite Echinococcus multilocularis is composed largely of carbohydrates, which form a tight microfibrillar meshwork around the entire metacestode. Since this laminated layer is the only parasite structure which is in constant contact with host immune and non-immune cells, and appears largely resistant to physiological and immunological reactions of the host, it most likely carries out important functions with regard to host–parasite interactions. In infected hosts, the metacestode is usually concentrically covered by host connective tissue cells and large amounts of collagen, causing a dense scar-like fibrosis, and it is likely that host-derived components are incorporated into the laminated layer at the host–parasite interface. Therefore, in order to obtain information on the molecular composition of this structure, we used parasite larvae which were generated through in vitro cultivation and thus were largely devoid of interfering host components. Lectin fluorescence on section-labelling of metacestodes embedded in LR-White suggested that the laminated layer is largely composed of N-acetyl- β- d-galactosaminyl, and α- and β- d-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine- β-1.3-galactose, while N-linked glycopeptides and α- d-mannosyl residues and/or glucosyl residues were found mainly within the germinal layer, and within the cellular mass and the surface of developing protoscoleces. Lectin–gold EM confirmed these findings. The laminated layer was isolated from in vitro cultivated metacestodes by urea extraction, and the ultrastructure of the purified laminated layer was assessed comparatively with respect to the laminated layer of intact parasites. The glycan composition was determined using SDS–PAGE and lectin blotting. This work has laid the basis for a more detailed dissection of the molecular composition of the laminated layer.