Vasa-Like DEAD-Box RNA Helicases of Schistosoma mansoni

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

An Atlas for Schistosoma mansoni Organs and Life-Cycle Stages Using Cell Type-Specific Markers and Confocal Microscopy

Schistosomiasis (bilharzia) is a tropical disease caused by trematode parasites (Schistosoma) that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel) is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites

Transcriptional Analysis of a Unique Set of Genes Involved in <em>Schistosoma mansoni</em> Female Reproductive Biology

<div><p>Schistosomiasis affects more than 200 million people globally. The pathology of schistosome infections is due to chronic tissue inflammation and damage from immune generated granulomas surrounding parasite eggs trapped in host tissues. <em>Schistosoma</em> species are unique among trematode parasites because they are dioecious; females require paring with male parasites in order to attain reproductive maturity and produce viable eggs. <em>Ex vivo</em> cultured females lose the ability to produce viable eggs due to an involution of the vitellarium and loss of mature oocytes. In order to better understand schistosome reproductive biology we used data generated by serial analysis of gene expression (SAGE) to identify uncharacterized genes which have different transcript abundance in mature females, those that have been paired with males, and immature females obtained from unisexual infections. To characterize these genes we used bioinformatics, transcript localization, and transcriptional analysis during the regression of <em>in vitro</em> cultured females. Genes transcribed exclusively in mature females localize primarily in the vitellocytes and/or the ovary. Genes transcribed exclusively in females from single sex infections localize to vitellocytes and subtegumental cells. As female reproductive tissues regress, eggshell precursor proteins and genes involved in eggshell synthesis largely have decreased transcript abundance. However, some genes with elevated transcript abundance in mature adults have increased gene expression following regression indicating that the genes in this study function both in eggshell biology as well as vitellogenesis and maintenance of female reproductive tissues. In addition, we found that genes enriched in females from single sex infections have increased expression during regression in <em>ex vivo</em> females. By using these transcriptional analyses we can direct research to examine the areas of female biology that are both relevant to understanding the overall process of female development and worm pairing while determining novel therapeutic approaches directed at the maturation of female schistosomes.</p> </div

WISH analysis of genes differentially transcribed in mature adult females.

<p>Whole-mount <i>in situ</i> hybridizations with riboprobes specific to each gene are shown. Panel A: p14; PanelB: FsMucin; Panel C: Fs800; Panel D: 10688; Panel E: 10548; Panel F: 11223; Panel G: 21110; Panel H: 11779; Panel I: 28488; Panel J: 10763; Panel K: 10401; Panel L: 21733; Panel M: 33844; Panel N: 10927; Panel O: 10403; Panel P: 8056; Panel Q: 8987; and Panel R: 10617, Panel S: 10435; Panel T: 11088; Panel U: 11283; Panel V:, 11055/CPEB1; Panel W: CPEB2; Panels X and Y: cgh-1, the boxed region in (X) is shown at higher magnification in (Y). Tissues indicated are vitellarium (arrowhead), the vitelline duct and the ovo-vitelline duct (black arrow), and the ovary (*). All images are representative of >30 female worms. Scale bars are 100 µm for all panels except Y (50 µm). Images were taken on a Zeiss AxioStar Plus and analyzed with ImageJ software.</p

6767 is a MEG-2 Family member.

<p>Panel A: Alignment of MEG-2 family members. Proteins include an ESP-15 (CAZ37453.2) and 6767, a MEG-2 family member that is enriched in females from single sex infections. Alignments were generated using ClustalW and Boxshade. Panel B: Exon map of 6767 on <i>S. mansoni</i> genome fragment SC_0319. Exons are designated a-l, sizes of exons are listed below exons.</p

WISH analysis of AFSS transcripts in female worms.

<p>Panel A: 6767 localized in AFSS worms. Panel B: higher magnification view of the boxed area in image A. 6767 localizes to vitellocytes (arrowhead) and subtegumental cytons (arrow). Panel C: 15402 localized in AFSS worms. Panel D: higher magnification view of the boxed area in image C. 15402 localizes in a punctate staining pattern in subtegumental cytons (arrows in D). Panel E: 6767 localized in AFMS worms following <i>in vitro</i> culture for 3 days. Panel F: higher magnification view of the boxed area in image E. 6767 transcripts are detected in the ovo-vitelline duct (red arrows). Panel G: 15402 localized in AFMS worms following <i>in vitro</i> culture for 3 days. Panel H: higher magnification view of the boxed area in image G. 15402 transcripts are detected in the ovo-vitelline duct (red arrows) and in a punctate staining pattern surrounding the ootype (red arrowhead). Scale bars are 100 µm in A,C, E, F and G and 50 µM in B, D, and H. All images generated with a Zeiss AxioStar Plus microscope.</p

Alignment of TES domain containing proteins.

<p>The signal peptide domains are overlined with a solid line, while the TES domain is overlined with a dashed line. Alignments were generated using ClustalW and BoxShade. Residues in black are identical in highlighted proteins while residues in grey are conservative changes.</p

Agarose gel electrophoresis of reverse transcriptase PCR of control transcripts, single sex female specific transcripts, and mature female enriched transcripts.

<p>The same cDNA was used for the β-tubulin control and the gene specific PCR reactions. Lanes are: 1: mature female, 2: single sex female, 3: mature male, 4: single sex male, 5: liver stage parasites. All gels shown are normalized to 1 µg of cDNA and β-tubulin controls. The image shown is a composite of multiple agarose gels.</p