Molecular interactions of proteins associated with surface membrane biogenesis and renewal in the tegument of Schistosoma mansoni

Abstract

The tegument of Schistosoma mansoni serves a variety of critical biological roles for the parasite, including uptake of nutrients, osmoregulation, immune evasion and immunomodulation. Dynein light chains (DLCs) and tetraspanins (TSPs) are prominent proteins in the tegument and appear to be intimately linked with other molecules present in the apical, host-interactive, membrane of the parasites. In other eukaryotic cells, cytoplasmic DLCs are a subunit of motor complexes involved in such diverse cellular translocation events as protein trafficking, mitosis and ciliary beating. DLCs of the schistosome tegument likely have similar roles and are likely to be important in the development and renewal of the tegument membrane, thereby contributing to parasite survival in its host. There is evidence that S. mansoni DLCs bind to a group of proteins unique to the tegument, the tegument allergen-like antigens (TALs) suggesting that schistosome DLCs may form complexes outside of the dynein complex. TSPs are membrane-spanning proteins that act as scaffolds for the formation of membraneassociated protein complexes comprising a wide variety of proteins. These TSP-enriched microdomains are known to play a wide range of roles within human cells, including maintenance of cell morphology and cell signalling.The biology of DLCs, TALs and TSPs in schistosome tegument is investigated in this study. Two DLCs, SmDLC1 and SmDLC5, three TALs, Sm20.8, Sm21.7 and Sm22.6 along with SmTSP-2 are the focus of this study. The tegumental localisation and transcription throughout the schistosome life cycle are reported for each transcript. By indirect immunocytochemistry the localisation within the tegument of SmTSP-2, SmDLC1 and Sm22.6 was investigated using parasites prepared by high pressure freezing for electron microscopy and cryosubstitution in uranyl acetate in solvent. SmTSP-2 was strongly membrane associated but little labelling was associated with the apical tegument membranes. SmDLC1 labelling was abundant in the entire tegument cytoplasm, close to the apical tegument membranes, surface invaginations and on the membranes on some tegumental vesicles. Sm22.6 had a more diffuse localisation pattern across the cytoplasm and labelling was apparent close to the apical surface membranes. Dual labelling immunocytochemistry of SmDLC1 and Sm22.6 did not show labelling patterns that confirmed the interaction of these SmDLCs and SmTALs. The transcript abundance of SmDLCs and SmTALs were assessed in the schistosome life cycle by real-time PCR. A similar pattern was seen for most transcripts, with transcript abundance increasing as the parasite developed from free-living life cycle stages (egg, miracidia, cercariae) to parasitic ii stages (schistosomula and adults). The most striking exception to this pattern is Sm21.7, which has a consistently high abundance across all life cycle stages.Protein interactions in the tegument were explored using Blue Native polyacrylamide gel electrophoresis and protein crosslinkers, coupled with in-line liqui...