Abstract
Genome sequences are available for the human blood flukes, Schistosoma japonicum, S. mansoni and S. haematobium. Functional genomic approaches could aid in identifying the role and importance of these newly described schistosome genes. Transgenesis is established for functional genomics in model species, which can lead to gain- or loss-of-functions, facilitate vector-based RNA interference, and represents an effective forward genetics tool for insertional mutagenesis screens. Progress toward routine transgenesis in schistosomes might be expedited if germ cells could be reliably localized in cultured schistosomes. Vasa, a member of the ATP-dependent DEAD-box RNA helicase family, is a prototypic marker of primordial germ cells and the germ line in the Metazoa. Using bioinformatics, 33 putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome. Moreover, three of the helicases exhibited vasa-like sequences; phylogenetic analysis confirmed the three vasa-like genes—termed Smvlg1, Smvlg2, and Smvlg3—were members of the Vasa/PL10 DEAD-box subfamily. Transcripts encoding Smvlg1, Smvlg2, and Smvlg3 were cloned from cDNAs from mixed sex adult worms, and quantitative real time PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia, with strikingly high expression in the undeveloped egg. Whole mount in situ hybridization (WISH) analysis revealed that Smvlg1, Smvlg2 and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germ line transgenesis of schistosomes, etiologic agents of major neglected tropical diseases.
Highlights
Schistosomiasis is considered the most important of the human helminthiases in terms of morbidity and mortality
Database searches indicated that S. mansoni has at least 33 DEAD-box helicases
A DEAD-box helicase known as Vasa is a determinant in germ line segregation and maintenance
Summary
Schistosomiasis is considered the most important of the human helminthiases in terms of morbidity and mortality. Draft genome sequences of all three of the major schistosome species are available [4,5,6,7], underscoring a pressing need to develop functional genomic approaches to identify the role and importance of schistosome genes that might be targeted for development of novel anti-schistosomal interventions. Retrovirus-mediated transgenesis is an established functional genomics approach for model species, e.g. Murine leukemia virus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein (VSVG) mediates somatic transgenesis in S. mansoni [9], vertical transmission of integrated transgenes has not been reported in schistosomes. Germ line transgenesis might be more readily monitored if schistosome germ cells could be localized and tracked in cultured forms of this pathogen
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