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Abstract: Schistosoma mansoni leucine aminopeptidase (LAP) is thought to play a central role in hatching of the miracidium from the schistosome egg. We identified two discrete LAPs genes in the S. mansoni genome, and their orthologs in S. japonicum. The similarities in sequence and exon/intron structure of the two genes, LAP1 and LAP2, suggest that they arose by gene duplication and that this occurred before separation of the mansoni and japonicum lineages. The SmLAP1 and SmLAP2 genes have different expression patterns in diverse stages of the cycle; whereas both are equally expressed in the blood dwelling stages (schistosomules and adult), SmLAP2 expression was higher in free living larval (miracidia) and in parasitic intra-snail (sporocysts) stages. We investigated the role of each enzyme in hatching of schistosome eggs and the early stages of schistosome development by RNA interference (RNAi). Using RNAi, we observed marked and specific reduction of mRNAs, along with a loss of exopeptidase activity in soluble parasite extracts against the diagnostic substrate l-leucine-7-amido-4-methylcoumarin hydroxide. Strikingly, knockdown of either SmLAP1 or SmLAP2, or both together, was accompanied by ≥80% inhibition of hatching of schistosome eggs showing that both enzymes are important to the escape of miracidia from the egg. The methods employed here refine the utility of RNAi for functional genomics studies in helminth parasites and confirm these can be used to identify potential drug targets, in this case schistosome aminopeptidases. [Copyright &y& Elsevier]

The recent release of draft genome sequences of two of the major human schistosomes has underscored the pressing need to develop functional genomics approaches for these significant pathogens. The sequence information also makes feasible genome-scale investigation of transgene integration into schistosome chromosomes. Retrovirus-mediated transduction offers a means to establish transgenic lines of schistosomes, to elucidate schistosome gene function and expression, and to advance functional genomics approaches for these parasites. We investigated the utility of the Moloney murine leukemia retrovirus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) for the transduction of Schistosoma mansoni and delivery of reporter transgenes into schistosome chromosomes. Schistosomula were exposed to virions of VSVG-pseudotyped MLV, after which genomic DNA was extracted from the transduced schistosomes. Southern hybridization analysis indicated the presence of proviral MLV retrovirus in the transduced schistosomes. Fragments of the MLV transgene and flanking schistosome sequences recovered using an anchored PCR-based approach demonstrated definitively that somatic transgenesis of schistosome chromosomes had taken place and, moreover, revealed widespread retrovirus integration into schistosome chromosomes. More specifically, MLV transgenes had inserted in the vicinity of genes encoding immunophilin, zinc finger protein Sma-Zic, and others, as well as near the endogenous schistosome retrotransposons, the fugitive and SRI. Proviral integration of the MLV transgene appeared to exhibit primary sequence site specificity, targeting a gGATcc-like motif. Reporter luciferase transgene activity driven by the schistosome actin gene promoter was expressed in the tissues of transduced schistosomula and adult schistosomes. Luciferase activity appeared to be developmentally expressed in schistosomula with increased activity observed after 1 to 2 wk in culture. These findings indicate the utility of VSVG-pseudotyped MLV for transgenesis of S. mansoni, herald a tractable pathway forward toward germline transgenesis and functional genomics of parasitic helminths, and provide the basis for comparative molecular pathogenesis studies of chromosomal lesions arising from retroviral integration into human compared with schistosome chromosomes. [ABSTRACT FROM AUTHOR]

The transposon piggyBac from the genome of the cabbage looper moth Trichoplusia ni has been observed in the laboratory to jump into the genomes of key model and pathogenic eukaryote organisms including mosquitoes, planarians, human and other mammalian cells, and the malaria parasite Plasmodium falciparum. Introduction of exogenous transposons into schistosomes has not been reported but transposon-mediated transgenesis of schistosomes might supersede current methods for functional genomics of this important human pathogen. In the present study we examined whether the piggyBac transposon could deliver reporter transgenes into the genome of Schistosoma mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters was introduced along with 7-methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. The activity of the helper transposase mRNA was confirmed by Southern hybridization analysis of genomic DNA from the transformed schistosomes, and hybridization signals indicated that the piggyBac transposon had integrated into numerous sites within the parasite chromosomes, piggyBac integrations were recovered by retrotransposon-anchored PCR, revealing characteristic piggyBac TTAA footprints in the vicinity of the endogenous schistosome retrotransposons Boudicca, SR1, and SR2. This is the first report of chromosomal integration of a transgene and somatic transgenesis of this important human pathogen, in this instance accomplished by mobilization of the piggyBac transposon. [ABSTRACT FROM AUTHOR]

Schistosomes are considered the most important of the helminth parasites of humans in terms of morbidity and mortality. Schistosomes employ proteolytic enzymes to digest host hemoglobin from ingested human blood, including a cathepsin D-like, aspartic protease that is overexpressed in the gut of the adult female schistosome. Because of its key role in parasite nutrition, this enzyme represents a potential intervention target. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cathepsin D gene locus of Schistosoma mansoni. Using the cDNA encoding S. mansoni cathepsin D as a probe, we isolated several positive bacterial artificial chromosomes (BAC) from a BAC library that represents an ∼8-fold coverage of the schistosome genome. Sequencing of BAC clone 25_J_24 revealed that the cathepsin D gene locus was ∼13 kb in length, and included seven exons interrupted by six introns. The exons ranged in length from 49 to 294 bp, and the introns from 30 to 5025 bp. The genomic organization of schistosome cathepsin D was similar in sequence, structure and complexity to human cathepsin D, including to a greater or lesser extent the conservation of all six exon/intron boundaries of the schistosome gene. It was less similar to aspartic protease genes of the nematodes Caenorhabditis elegans and Haemonchus contortus, and dissimilar to those of plasmepsins from malarial parasites. Examination of the introns revealed the presence of endogenous mobile genetic elements including SR2, the ASL-associated retrotransposon, and the SINE-like element, SMα. Phylogenetically, schistosome cathepsin D appeared to be more closely related to mammalian cathepsin D than to other sub-families of eukaryotic aspartic proteases known from mammals. Taken together, these features indicated that schistosome cathepsin D is a platyhelminth orthologue of mammalian lysosomal cathepsin D. [Copyright &y& Elsevier]

Abstract: Retroviral transduction of cultured schistosomes offers a potential means to establish transgenic lines of schistosomes and thereby to facilitate the elucidation of schistosome gene function and expression. The Moloney murine leukemia retroviral (MMLV) vector pLNHX was modified to incorporate EGFP or luciferase reporter genes under control of schistosome endogenous gene promoters from the spliced leader RNA and HSP70 genes. These constructs and a plasmid encoding vesicular stomatitis virus glycoprotein (VSVG) were utilized along with GP2-293 cells to produce replication incompetent retrovirus particles pseudotyped with the VSVG envelope. Exposure of several developmental stages, including sporocysts, of Schistosoma mansoni to these virions was facilitated by incubation with polybrene and/or by centrifugation. The early stages of binding and uptake of virus to the parasite tegument were demonstrated by the immunofluorescence colocalization of VSVG envelope and retroviral capsid proteins. Southern hybridization analysis indicated the integration of proviral forms of the MMLV constructs in genomic DNA isolated from the virus exposed schistosomes. Furthermore, analysis of RNA isolated from virus treated parasites demonstrated the presence of transcripts encoding reporter transgenes. Together these results indicated productive transduction by VSVG pseudotyped MMLV of cultured schistosomes, and suggest a tractable route forward towards heritable schistosome transgenesis. [Copyright &y& Elsevier]