Schistosoma mansoni: germ-line transformation approaches and actin-promoter analysis.

Towards germ-line transformation miracidia were biolistically transformed with GFP reporter gene constructs and successfully reintroduced into the schistosome cycle. By PCR and confocal microscopy the presence and the expression of GFP were confirmed in cercariae or adults of the F(0) and F(1) generations. This indicated the presence of the constructs in the germ-line, although no evidence for genome integration was obtained. About 3kb of 5' upstream sequences of the actin gene SmAct1 were identified by in silico analyses, and different fragments up to 1.5kb subcloned for GFP-vector construction. A 445bp fragment was sufficient for transcription initiation in larvae or adults as confirmed by confocal microscopy. An actin gene characteristic assembly of TATA, CArG, and CAAT boxes has been identified, which seems to be functionally conserved between vertebrates and invertebrates. However, a vertebrate-specific intron containing an additional regulatory CArG box was not found indicating that the regulation of SmAct1 transcription depends exclusively on its promoter region.

Biolistic transformation of Schistosoma mansoni with 5' flanking regions of two peptidase genes promotes tissue-specific expression.

The gene-regulatory elements controlling peptidase expression in Schistosoma mansoni are unknown. A genomic DNA library was constructed from which 5' flanking fragments of the cathepsins F (SmCF; 649 bp) and B2 (SmCB2; 810 bp) peptidase genes were isolated. These were cloned into a GFP-expression vector for biolistic transformation of schistosomes. Both fragments promoted expression of GFP that was localised in the gut for SmCF and tegument for SmCB2, consistent with previous immunochemical data. Promoter-deletion of the SmCF gene indicated the importance of one or more transcription factor binding sites in the first 169 bp for both GFP-expression and its tissue specificity.

Transplantation of in vitro-generated Schistosoma mansoni mother sporocysts into Biomphalaria glabrata.

Specific studies on schistosome gene functions require both access to the parasite stages, preferably the larvae, and to complete the life cycle. In the present study, we investigated whether short-term in vitro cultivation of sporocysts and surgical transplantation into snails could be combined to produce cercariae. Miracidia were maintained in vitro in the presence of Biomphalaria glabrata embryonic (Bge) cells or, alternatively, in Bge-cell-conditioned medium. The transformation of miracidia to mother sporocysts was observed in both cases. Two day-old sporocysts were transplanted into the cephalopedal sinus of recipient snails. Transplantation efficiencies varied between 16% and 43%, depending on the culture of the sporocysts in terms of the number of cercariae producing snails. Cercariae recovered from these snails were used to successfully infect hamsters, demonstrating that short term in vitro-generated sporocysts undergo normal cercariogenesis following transplantation. This combination of in vitro cultivation and transplantation may be useful for novel experimental approaches to investigate the genes involved in larval development or host-parasite molecular interactions.

The uptake of Texas Red-BSA in the excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males.

The excretory system of schistosomes has focused some attention during the last years since accumulating evidence suggests that it plays an important role in the host-parasite interaction. Signalling molecules such as phosphatases, but also proteases have been localised in the excretory system. To some extent, however, localisation studies are limited by the fact that sections of fixed specimens are used. In this study, we tested the fluorescent molecules FITC-dextran and Texas Red-BSA for their ability to enter the excretory system of living Schistosoma mansoni males. It is demonstrated that the dyes selectively stain the excretory tubules which are widely distributed along the worm body. This finding was used to investigate whether the staining of worms with Texas Red-BSA can help to localise transgene activity in worms which were transiently transformed by particle bombardment. A vector was used for transformation which contained the green fluorescent protein gene, under the control of the regulatory elements of the cysteine protease ER60 gene. After transformation and staining, confocal laser scanning microscopy revealed that ER60-induced green fluorescent protein activity colocalises with Texas Red-BSA in the excretory tubules. The results suggest a role for ER60 during the host-parasite interaction. Furthermore, the colocalisation approach introduced here opens further perspectives to characterise gene-expression profiles in this parasite.

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.

HSP70-controlled GFP expression in transiently transformed schistosomes.

Among the parasitic helminths schistosomes are of high medical and economic importance. Despite of the world-wide relevance of this parasite, very little is known about the cellular mechanisms controlling its development and concerning the host-parasite interaction. Within the last decade a great effort has been made in this blood fluke to identify genes which play important roles during these processes. However, molecular analysis was limited by the fact, that neither function nor regulation of candidate genes could be investigated in this organism due to the lack of transformation protocols. Here, we present the strategy of ballistic gene transfer to introduce and characterize transgenes in different schistosome life stages. As a transformation vector, the heat shock protein 70 (hsp70) gene promoter and terminator from Schistosoma mansoni were cloned and fused to the green fluorescent protein (GFP) reporter gene. In a first attempt, the hsp70--GFP vector was successfully tested in a eukaryotic cell line. Thereafter, adult male schistosomes and sporocysts were transformed with this vector, and GFP expression was demonstrated using molecular and microscopical methods. PCR, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analyses confirmed the presence, transcription and translation of the transgene in adults. Confocal laser scanning microscopy revealed GFP-activity at various sites along the surface of the worms after hs induction and within sporocysts. These results suggest diverse roles for hsp70 during the development of schistosomes. Furthermore, the results demonstrate the feasibility of this method and open the perspective to analyze a variety of molecular functions in schistosomes.