Characterization of Vasa-like Genes of Schistosoma mansoni, Including Functional Genomic Analyses
Open AccessGenome sequences are available for each of the major human blood flukes, Schistosoma japonicum , S. mansoni , and S. haematobium . Functional genomic approaches could aid in identifying the role and importance of 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 towards 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 germline in the Metazoa. Vasa-like sequences are known from other platyhelminths including the monogenean Neobendenia girellae, a representative parasitic flatworm, and planarians. The central hypothesis underpinning this dissertation is that the schistosome genome encodes a vasa-like gene orthologue(s). It was hypothesized further that the postulated schistosome vasa-like gene(s) perform roles in the development of S. mansoni (representative of the human schistosomes at large) similar to roles described in other flatworms. To test these hypotheses, the following specific aims were explored: 1. Identification of S. mansoni vasa-like gene(s) within the schistosome genome, assisted by bioinformatics approaches; 2. Expression analysis of S. mansoni vasa-like gene(s) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and whole mount in situ hybridization (WISH); 3. Functional analysis of the S. mansoni vasa-like gene(s) by RNA interference; and 4. Investigation of impact of gene silencing of schistosome vasa-like gene(s) on retrovirus based transgenesis of S. mansoni.Thirty-three putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome using bioinformatic approaches. Three of the helicases exhibited vasa-like sequences, and 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 S. mansoni worms, and qRT-PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia. Strikingly high expression was observed in the undeveloped egg. WISH analysis revealed that Smvlg1, Smvlg2, and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Developmental expression profiles of Smvlg1, Smvlg2, and Smvlg3 were similar--each gene was expressed in all developmental stages of S. mansoni examined. One explanation for why the transcript expression of the Smvlgs is in every stage of the schistosome is that they may be expressed in the cells that continue the germinal lineage of the schistosome.RNA interference was performed to decipher the function(s) of these vasa-like genes. After delivery of dsRNA, the morphology of the ovary in adult females was investigated. Introduction of dsRNA targeting Smvlg1 by electroporation reduced the volume of the ovaries in adult female worms. This morphological change in the ovaries of treated schistosomes was detected with a novel technique using carmine red-staining of the parasites followed by confocal laser scanning microscopy (CLSM) and interpretation of the images with the assistance of the Velocity software package. Reduced ovary volume evident after gene silencing of one of the vasa-like genes, i.e Smvlg1, may have been caused by RNAi-induced loss of germ cells. This suggested that Smvlg1 was essential to germ cells in the schistosome ovary. By contrast, significant differences were not apparent in number of eggs produced or hatching rate of eggs laid by female schistosomes treated with Smvlg-specific dsRNAs. Together these findings suggested that Smvlg1 was essential for development of the schistosome ovary. A practical use for these vasa-like genes was investigated; specifically, the impact of gene silencing of Smvlg1, Smvlg2, and Smvlg3 on the performance of chromosomal integration of retroviral transgenes. The philosophy underpinning this approach was that since homeostatic silencing by RNA interference is central to regulating mobilization of endogenous transposable elements (TEs), and fundamental to protection of the germline from uncontrolled mobilization of TEs, silencing of vasa-like genes may be a tractable method to improve experimental, TE-based transgenesis of schistosomes. Triplicate experiments were carried out that included silencing of the vasa-like genes, transduction of the gene-silenced worms with pseudotyped retroviral (murine leukemia virus) virions, and quantitative retrotransposon anchored PCR (qRAP) to ascertain levels of transcription of a reporter transgene (neoR). Whereas neoR levels were elevated, compared to control, indicating that silencing of the vasa-like genes had facilitated retroviral integration into the schistosome chromosomes, the PCR based findings were not reliably reproduced. In overview, the vasa /PL10-like gene Smvlg1 appears to be involved in germ cell development in the female schistosome, although this function/role awaits deeper investigation. In addition, the newly described germline specific role of Smvlg1, together with specific expression in the ovary of S. mansoni predicts that it has utility as a germline specific marker for oogenesis, germline transgenesis, and gametogenesis. Lastly, this study established a new technique for measuring the volume of the schistosome ovary, a method that will likely find wider use in functional genomics of schistosomes and other platyhelminth parasites. To conclude, germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germline transgenesis of schistosomes, etiologic agents of major neglected tropical diseases.
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