The growing availability of sequence information from diverse parasites through genomic and transcriptomic projects offer new opportunities for the identification of key mediators in the parasite–host interaction. Functional genomics approaches and methods for the manipulation of genes are essential tools for deciphering the roles of genes and to identify new intervention targets in parasites. Exciting advances in functional genomics for parasitic helminths are starting to occur, with transgene expression and RNA interference (RNAi) reported in several species of nematodes, but the area is still in its infancy in flatworms, with reports in just three species. While advancing in model organisms, there is a need to rapidly extend these technologies to other parasites responsible for several chronic diseases of humans and cattle. In order to extend these approaches to less well studied parasitic worms, we developed a test method for the presence of a viable RNAi pathway by silencing the exogenous reporter gene, firefly luciferase (fLUC). We established the method in the human blood fluke Schistosoma mansoni and then confirmed its utility in the liver fluke Fasciola hepatica. We transformed newly excysted juveniles of F. hepatica by electroporation with mRNA of fLUC and three hours later were able to detect luciferase enzyme activity, concentrated mainly in the digestive ceca. Subsequently, we tested the presence of an active RNAi pathway in F. hepatica by knocking down the exogenous luciferase activity by introduction into the transformed parasites of double-stranded RNA (dsRNA) specific for fLUC. In addition, we tested the RNAi pathway targeting an endogenous F. hepatica gene encoding leucine aminopeptidase (FhLAP), and observed a significant reduction in specific mRNA levels. In summary, these studies demonstrated the utility of RNAi targeting reporter fLUC as a reporter gene assay to establish the presence of an intact RNAi pathway in helminth parasites. These could facilitate the study of gene function and the identification of relevant targets for intervention in organisms that are by other means intractable. More specifically, these results open new perspectives for functional genomics of F. hepatica, which hopefully can lead to the development of new interventions for fascioliasis.
References
[1]
Kalinna BH, Brindley PJ (2007) Manipulating the manipulators: advances in parasitic helminth transgenesis and RNAi. Trends Parasitol 23: 197–204. doi: 10.1016/j.pt.2007.03.007
[2]
Brindley PJ, Pearce EJ (2007) Genetic manipulation of schistosomes. Int J Parasitol 37: 465–473. doi: 10.1016/j.ijpara.2006.12.012
[3]
Wilson RA, Ashton PD, Braschi S, Dillon GP, Berriman M, et al. (2007) 'Oming in on schistosomes: prospects and limitations for post-genomics. Trends Parasitol 23: 14–20. doi: 10.1016/j.pt.2006.10.002
[4]
Geldhof P, Visser A, Clark D, Saunders G, Britton C, et al. (2007) RNA interference in parasitic helminths: current situation, potential pitfalls and future prospects. Parasitology 134: 609–619. doi: 10.1017/S0031182006002071
[5]
Wippersteg V, Kapp K, Kunz W, Grevelding CG (2002) Characterisation of the cysteine protease ER60 in transgenic Schistosoma mansoni larvae. Int J Parasitol 32: 1219–1224. doi: 10.1016/S0020-7519(02)00092-9
Wippersteg V, Ribeiro F, Liedtke S, Kusel JR, Grevelding CG (2003) The uptake of Texas Red-BSA in the excretory system of schistosomes and its colocalisation with ER60 promoter-induced GFP in transiently transformed adult males. Int J Parasitol 33: 1139–1143. doi: 10.1016/S0020-7519(03)00168-1
[8]
Wippersteg V, Sajid M, Walshe D, Khiem D, Salter JP, et al. (2005) Biolistic transformation of Schistosoma mansoni with 5′ flanking regions of two peptidase genes promotes tissue-specific expression. Int J Parasitol 35: 583–589. doi: 10.1016/j.ijpara.2005.02.002
[9]
Davis RE, Parra A, LoVerde PT, Ribeiro E, Glorioso G, et al. (1999) Transient expression of DNA and RNA in parasitic helminths by using particle bombardment. Proc Natl Acad Sci U S A 96: 8687–8692. doi: 10.1073/pnas.96.15.8687
[10]
Heyers O, Walduck AK, Brindley PJ, Bleiss W, Lucius R, et al. (2003) Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts. Exp Parasitol 105: 174–178. doi: 10.1016/j.exppara.2003.11.001
[11]
Rossi A, Wippersteg V, Klinkert MQ, Grevelding CG (2003) Cloning of 5′ and 3′ flanking regions of the Schistosoma mansoni calcineurin A gene and their characterization in transiently transformed parasites. Mol Biochem Parasitol 130: 133–138. doi: 10.1016/S0166-6851(03)00158-0
[12]
Correnti JM, Pearce EJ (2004) Transgene expression in Schistosoma mansoni: introduction of RNA into schistosomula by electroporation. Mol Biochem Parasitol 137: 75–79. doi: 10.1016/j.molbiopara.2004.04.015
[13]
Yuan XS, Shen JL, Wang XL, Wu XS, Liu DP, et al. (2005) Schistosoma japonicum: a method for transformation by electroporation. Exp Parasitol 111: 244–249. doi: 10.1016/j.exppara.2005.08.010
[14]
Morales ME, Mann VH, Kines KJ, Gobert GN, Fraser MJ Jr., et al. (2007) piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni. Faseb J.. doi: 10.1096/fj.07-8726com
[15]
Kines KJ, Mann VH, Morales ME, Shelby BD, Kalinna BH, et al. (2006) Transduction of Schistosoma mansoni by vesicular stomatitis virus glycoprotein-pseudotyped Moloney murine leukemia retrovirus. Exp Parasitol 112: 209–220. doi: 10.1016/j.exppara.2006.02.003
[16]
Skelly PJ, Da'dara A, Harn DA (2003) Suppression of cathepsin B expression in Schistosoma mansoni by RNA interference. Int J Parasitol 33: 363–369. doi: 10.1016/S0020-7519(03)00030-4
[17]
Boyle JP, Wu XJ, Shoemaker CB, Yoshino TP (2003) Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts. Mol Biochem Parasitol 128: 205–215. doi: 10.1016/S0166-6851(03)00078-1
[18]
Correnti JM, Brindley PJ, Pearce EJ (2005) Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth. Mol Biochem Parasitol 143: 209–215. doi: 10.1016/j.molbiopara.2005.06.007
[19]
Osman A, Niles EG, Verjovski-Almeida S, LoVerde PT (2006) Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene. PLoS Pathog 2: e54. doi:10.1371/journal.ppat.0020054. doi: 10.1371/journal.ppat.0020054
[20]
Krautz-Peterson G, Radwanska M, Ndegwa D, Shoemaker CB, Skelly PJ (2007) Optimizing gene suppression in schistosomes using RNA interference. Mol Biochem Parasitol 153: 194–202. doi: 10.1016/j.molbiopara.2007.03.006
[21]
Cheng GF, Lin JJ, Shi Y, Jin YX, Fu ZQ, et al. (2005) Dose-dependent inhibition of gynecophoral canal protein gene expression in vitro in the schistosome (Schistosoma japonicum) by RNA interference. Acta Biochim Biophys Sin (Shanghai) 37: 386–390. doi: 10.1111/j.1745-7270.2005.00058.x
[22]
Dinguirard N, Yoshino TP (2006) Potential role of a CD36-like class B scavenger receptor in the binding of modified low-density lipoprotein (acLDL) to the tegumental surface of Schistosoma mansoni sporocysts. Mol Biochem Parasitol 146: 219–230. doi: 10.1016/j.molbiopara.2005.12.010
[23]
Delcroix M, Sajid M, Caffrey CR, Lim KC, Dvorak J, et al. (2006) A multienzyme network functions in intestinal protein digestion by a platyhelminth parasite. J Biol Chem 281: 39316–39329. doi: 10.1074/jbc.M607128200
[24]
Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sanchez Alvarado A (2005) Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev Cell 8: 635–649. doi: 10.1016/j.devcel.2005.02.014
[25]
Newmark PA (2005) Opening a new can of worms: a large-scale RNAi screen in planarians. Dev Cell 8: 623–624. doi: 10.1016/j.devcel.2005.04.007
[26]
Newmark PA, Reddien PW, Cebria F, Sanchez Alvarado A (2003) Ingestion of bacterially expressed double-stranded RNA inhibits gene expression in planarians. Proc Natl Acad Sci U S A 100: Suppl 111861–11865. doi: 10.1073/pnas.1834205100
[27]
Sanchez Alvarado A (2003) The freshwater planarian Schmidtea mediterranea: embryogenesis, stem cells and regeneration. Curr Opin Genet Dev 13: 438–444. doi: 10.1016/S0959-437X(03)00082-0
[28]
Haas BJ, Berriman M, Hirai H, Cerqueira GG, Loverde PT, et al. (2007) Schistosoma mansoni genome: Closing in on a final gene set. Exp Parasitol. doi: 10.1016/j.exppara.2007.06.005
[29]
Liu F, Lu J, Hu W, Wang SY, Cui SJ, et al. (2006) New perspectives on host-parasite interplay by comparative transcriptomic and proteomic analyses of Schistosoma japonicum. PLoS Pathog 2: e29. doi:10.1371/journal.ppat.0020029. doi: 10.1371/journal.ppat.0020029
[30]
Verjovski-Almeida S, DeMarco R, Martins EA, Guimaraes PE, Ojopi EP, et al. (2003) Transcriptome analysis of the acoelomate human parasite Schistosoma mansoni. Nat Genet 35: 148–157. doi: 10.1038/ng1237
[31]
Verjovski-Almeida S, Leite LC, Dias-Neto E, Menck CF, Wilson RA (2004) Schistosome transcriptome: insights and perspectives for functional genomics. Trends Parasitol 20: 304–308. doi: 10.1016/j.pt.2004.04.012
[32]
Hu W, Yan Q, Shen DK, Liu F, Zhu ZD, et al. (2003) Evolutionary and biomedical implications of a Schistosoma japonicum complementary DNA resource. Nat Genet 35: 139–147. doi: 10.1038/ng1236
[33]
Spiliotis M, Lechner S, Tappe D, Scheller C, Krohne G, et al. (2007) Transient transfection of Echinococcus multilocularis primary cells and complete in vitro regeneration of metacestode vesicles. Int J Parasitol. doi: 10.1016/j.ijpara.2007.11.002
[34]
Ohashi H, Umeda N, Hirazawa N, Ozaki Y, Miura C, et al. (2007) Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae. Int J Parasitol 37: 515–523. doi: 10.1016/j.ijpara.2006.11.003
[35]
McGonigle L, Mousley A, Marks NJ, Brennan GP, Dalton JP, et al. (2008) The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration. Int J Parasitol 38: 149–155. doi: 10.1016/j.ijpara.2007.10.007
[36]
Spithill TW, Smooker PM, Copeman B (1999) Fasciola gigantica: epidemiology, control, immunology and molecular biology. In: Dalton JP, editor. Fasciolosis. Wallingford: CAB International Publishing. pp. 465–525.
[37]
Mas-Coma S (2005) Epidemiology of fascioliasis in human endemic areas. J Helminthol 79: 207–216. doi: 10.1079/JOH2005296
Keiser J, Engels D, Buscher G, Utzinger J (2005) Triclabendazole for the treatment of fascioliasis and paragonimiasis. Expert Opin Investig Drugs 14: 1513–1526. doi: 10.1517/13543784.14.12.1513
[40]
Brennan GP, Fairweather I, Trudgett A, Hoey E, McCoy , et al. (2007) Understanding triclabendazole resistance. Exp Mol Pathol 82: 104–109. doi: 10.1016/j.yexmp.2007.01.009
Morphew RM, Wright HA, LaCourse EJ, Woods DJ, Brophy PM (2007) Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host. Mol Cell Proteomics 6: 963–972. doi: 10.1074/mcp.M600375-MCP200
[43]
Lazdins JK, Stein MJ, David JR, Sher A (1982) Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll. Exp Parasitol 53: 39–44. doi: 10.1016/0014-4894(82)90090-X
[44]
Basch PF (1981) Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. J Parasitol 67: 179–185. doi: 10.2307/3280632
[45]
Carmona C, Dowd AJ, Smith AM, Dalton JP (1993) Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Mol Biochem Parasitol 62: 9–17. doi: 10.1016/0166-6851(93)90172-T
[46]
Acosta D, Cancela M, Piacenza L, Roche L, Carmona C, et al. (2007) Fasciola hepatica leucine aminopeptidase, a promising candidate for vaccination against ruminant fasciolosis. Mol Biochem Parasitol. doi:10.1016/j.molbiopara.2007.11.011.
[47]
Correnti JM, Jung E, Freitas TC, Pearce EJ (2007) Transfection of Schistosoma mansoni by electroporation and the description of a new promoter sequence for transgene expression. Int J Parasitol 37: 1107–1115. doi: 10.1016/j.ijpara.2007.02.011
[48]
Smith MA, Clegg JA (1981) Improved culture of Fasciola hepatica in vitro. Z Parasitenkd 66: 9–15. doi: 10.1007/BF00941940
[49]
Fairweather I, Threadgold LT, Hanna REB (1999) Development of Fasciola hepatica in the mammalian host. In: Dalton JP, editor. Fasciolosis. New York: CABI Publishing. pp. 47–104.
[50]
Morales ME, Rinaldi G, Gobert GN, Kines KJ, Tort JF, et al. (2007) RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade. Mol Biochem Parasitol. doi:10.1016/j.molbiopara.2007.10.009.
[51]
Cancela M, Acosta D, Rinaldi G, Silva E, Duran R, et al. (2008) A distinctive repertoire of cathepsins is expressed by juvenile invasive Fasciola hepatica. Biochimie Accepted. doi: 10.1016/j.biochi.2008.04.020
[52]
Knox DP, Geldhof P, Visser A, Britton C (2007) RNA interference in parasitic nematodes of animals: a reality check? Trends Parasitol 23: 105–107. doi: 10.1016/j.pt.2007.01.007
[53]
Viney ME, Thompson FJ (2007) Two hypotheses to explain why RNA interference does not work in animal parasitic nematodes. Int J Parasitol. doi: 10.1016/j.ijpara.2007.08.006
[54]
Winston WM, Sutherlin M, Wright AJ, Feinberg EH, Hunter CP (2007) Caenorhabditis elegans SID-2 is required for environmental RNA interference. Proc Natl Acad Sci U S A 104: 10565–10570. doi: 10.1073/pnas.0611282104
[55]
Ghedin E, Wang S, Spiro D, Caler E, Zhao Q, et al. (2007) Draft genome of the filarial nematode parasite Brugia malayi. Science 317: 1756–1760. doi: 10.1126/science.1145406
