Characterisation of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae.
Proteinases have been found to play important roles in parasites. They are involved in developmental processes and facilitate invasion of host tissues as well as the digestion of host molecules for nutrition. The cysteine protease ER60 from Schistosoma mansoni, originally characterised in adults to be expressed in excretory organs, was analysed in larval stages. Transcripts were found in miracidia, in vitro generated mother sporocysts and cercariae. After cloning the promoter and terminator of the ER60 gene, a transformation vector was constructed containing the green fluorescent protein reporter gene flanked by the regulatory elements. The ER60-green fluorescent protein vector was used for transfection experiments of COS-7 cells demonstrating the functionality of the promoter in the heterologous system. To analyse the expression pattern of ER60-green fluorescent protein in larval S. mansoni, in vitro generated mother sporocysts were transformed by particle bombardment, a method which allows gene transfer into schistosomes. Molecular analyses demonstrated transcription and translation of the transgene. Furthermore, confocal laser scanning microscopy revealed ER60-induced green fluorescent protein fluorescence within the larvae. Inside primary sporocysts, tissue-specific activity was localised in the gland cells, protonephridia and several cytons. These results suggest that ER60 is expressed in the ES system of larvae and, amongst other functions, may play a role in penetration and migration processes.
Institute for Genetics, Genetic Parasitology and Center for Biological and Medical Research, Heinrich-Heine-University, D-40225 Düsseldorf, Germany.
Volker
V
Wippersteg
Katja
K
Kapp
Werner
W
Kunz
Christoph G
CG
Grevelding
D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000032:analysis*; D015801:Helminth Proteins / Q000032:analysis*; D007814:Larva / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D008018:Life Cycle Stages; D008957:Models, Genetic; D008969:Molecular Sequence Data; D033661:Oocysts / Q000235:genetics / Q000528:radiation effects; D012550:Schistosoma mansoni / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods
D015801:Helminth Proteins; D003546:Cysteine Endopeptidases