Gene knockdown in Schistosoma mansoni Targeting the Hemoglobin Proteolysis Cascade

Functional genomics tools are needed for schistosomes. This dissertation deals with the development of vector based RNA interference (RNAi) for Schistosoma mansoni, with a focus on construction and testing of plasmid driven short hairpin RNAs in schistosomes, targeting informative, model genes. The reporter firefly luciferase is known to be tractable and active in schistosome tissues. The aspartic protease cathepsin D was targeted since it is a well characterized schistosome gene known to be amenable to genetic manipulation. Cathepsin D is expressed in the schistosome gut where it digests hemoglobin released from ingested host erythrocytes. RNAi was employed to investigate gene silencing using short interfering RNA (siRNA) and longer double stranded RNA (dsRNA) specific for cathepsin D transcript. 48 hours old schistosomules were exposed to 30 mug dsRNA or 10 mug of short interfering RNAs (siRNA) specific for discrete sites on the target cDNA. siRNAs constructs were made to target three different locations on the alpha, beta and gamma. The alpha- and gamma-siRNAs delivered stronger knockdown than the beta-siRNA. To develop a vector based RNAi model for S. mansoni, the schistosome U6 gene promoter was employed to drive expression of shRNA targeting reporter firefly luciferase. An upstream region of a U6 gene predicted to contain the promoter was amplified from genomic DNA of S. mansoni . A shRNA construct driven by the predicted U6 promoter targeting luciferase was assembled and cloned into the piggyBac plasmid pXL-Bac II, the construct termed pXL-BacII_SmU6-shLuc. Luciferase expression in transgenic fibrosarcoma HT-1080 cells was significantly reduced 96 hours following transfection with plasmid PXL-BacII_SmU6-shLuc, which encodes luciferase mRNA-specific shRNA. In like fashion, schistosomules of S. mansoni were transformed with the SmU6-shLuc or control constructs. Firefly luciferase mRNA was introduced into transformed schistosomules after which luciferase activity was analyzed. Significantly less activity was present in schistosomules transfected with pXL-BacII_SmU6-shLuc compared to controls. These findings revealed that the putative S. mansoni U6 gene promoter of 270 bp in length was active in human cells and schistosomes, and in like fashion a plasmid termed pXL-BacII_HsU6-shLuc also drove shRNA targeting luciferase in schistosomes. Remarkably, when employed in vector based RNAi studies in schistosomes, pXL-BacII_HsU6-shLuc was more efficient in silencing firefly luciferase than was PXL-BacII_SmU6-shLuc.