Bedeutung von Protein-Glykan-Wechselwirkungen bei der Pathogenese von Myxobolus cerebralis, dem Erreger der Drehkrankheit der Salmoniden

摘要

Glycans and sugar binding molecules (lectins) form a mutual recognition system which enables parasitic organisms to adhere on host cells and to migrate to target tissue. The aim of the present study was to detect and localize carbohydrate-binding activities in developmental stages of M. cerebralis. The binding patterns of thirteen biotin-labelled neoglycoconjugates were examined histochemically in thin-sections of infected rainbow trout (Oncorhynchus mykiss) and oligochaete (Tubifex tubifex) and preparations of waterborne triactinomyxon spores. A further aim was to characterise glycan epitopes in the myxospore stage of M. cerebralis. Lectin and Western blotting analyses were performed by using selected biotin-labelled plant lectins (GSA-I, PHA-E, SJA, GSA-II) and TAM-antiserum. Glycoproteins were isolated by using lectin affinity chromatography and prominent bands were characterised by mass spectrometry (MALDI/MS). Distinct structure-selective and developmental stage-regulated expression of certain classes of carbohydrate binding was observed. In TAM spores, expression of carbohydrate binding activity specific for alpha-L-Fuc-BSA-biotin, alpha-D-GalNAc-BSA-biotin, Lac-BSA-biotin und ASF-biotin was up-regulated in the polar capsules; shell valves showed no activity. In the gut of T. tubifex, polar capsules of the parasite showed strong positive reaction only with beta-D-GlcNAc-BSA-biotin. In fish cartilage polar capsules were negative, but the spore shell valves showed a broad range of carbohydrate binding activities. No activity was detected for either alpha 2-6- or alpha 2-3-linked N-acetyl-D-neuraminic acid. An adhesion assay was performed and myxospores were found to specifically adhere to matrices containing residues of lactose, fucose, galactose, N-acetyl-D-galactosamine and N-acetyl-D-glucosamine. Furthermore it was identified that glycoepitopes of myxospores contain carbohydrate motifs reactive with PHA-E (proteins 7-70 kDa), SJA (7-70 kDa), GSA-I (10-209 kDa) and GSA-II (5-40 kDa). Mcgp33, a glycoprotein isolated by lectin affinity chromatography is reactive with SJA (about 33 kDa) and is exclusively expressed in the myxospore stage. The application of antiserum against triactinomyxon spores of M. cerebralis indicates differences in antigenicity of isolated glycoproteins from both, waterborne TAM spores and M. cerebralis myxospores. This is the first investigation which identifies lectin activity in a parasite of the phylum myxozoa. A participation of the characterised molecules is suspected in the parasite to mediate the complex process of host recognition, adhesion and invasion. Furthermore, the detected activities can participate in the protection of the spores against environmental forces and may generate immune reactions during migration in the host tissue.