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Silencing of essential genes by RNA interference in Haemonchus contortus

  • J. L. ZAWADZKI (a1), A. C. KOTZE (a2), J.-A. FRITZ (a3), N. M. JOHNSON (a3), J. E. HEMSWORTH (a1), B. M. HINES (a2) and C. A. BEHM (a3)...

Summary

In this study we assessed three technologies for silencing gene expression by RNA interference (RNAi) in the sheep parasitic nematode Haemonchus contortus. We chose as targets five genes that are essential in Caenorhabditis elegans (mitr-1, pat-12, vha-19, glf-1 and noah-1), orthologues of which are present and expressed in H. contortus, plus four genes previously tested by RNAi in H. contortus (ubiquitin, tubulin, paramyosin, tropomyosin). To introduce double-stranded RNA (dsRNA) into the nematodes we tested (1) feeding free-living stages of H. contortus with Escherichia coli that express dsRNA targetting the test genes; (2) electroporation of dsRNA into H. contortus eggs or larvae; and (3) soaking adult H. contortus in dsRNA. For each gene tested we observed reduced levels of mRNA in the treated nematodes, except for some electroporation conditions. We did not observe any phenotypic changes in the worms in the electroporation or dsRNA soaking experiments. The feeding method, however, elicited observable changes in the development and viability of larvae for five of the eight genes tested, including the ‘essential’ genes, Hc-pat-12, Hc-vha-19 and Hc-glf-1. We recommend the E. coli feeding method for RNAi in H. contortus and provide recommendations for future research directions for RNAi in this species.

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Corresponding author

*Corresponding author: Division of Biomedical Science and Biochemistry, Research School of Biology, The Australian National University, Canberra, A.C.T. 0200, Australia. Tel: +61 2 61252203. Fax: +61 2 61250313. E-mail: Carolyn.Behm@anu.edu.au

References

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Abdel-Wahab, N., Kuo, Y. M., Wu, Y., Tuan, R. S. and Bianco, A. E. (1996). OvB20, an Onchocerca volvulus-cloned antigen selected by differential immunoscreening with vaccination serum in a cattle model of onchocerciasis. Molecular and Biochemical Parasitology 76, 187199. doi: 10.1016/0166-6851(95)02558-8.
Albers, G. A. and Burgess, S. K. (1988). Serial passage of Haemonchus contortus in resistant and susceptible sheep. Veterinary Parasitology 28, 303306. doi: 10.1016/0304-4017(88)90077-5
Behm, C. A., Bendig, M. M., McCarter, J. P. and Sluder, A. E. (2005). RNAi-based discovery and validation of new drug targets in filarial nematodes. Trends in Parasitology 21, 97100. doi: 10.1016/j.pt.2004.12.003.
Berezikov, E., Bargmann, C. I. and Plasterk, R. H. A. (2004). Homologous gene targeting in Caenorhabditis elegans by biolistic transformation. Nucleic Acids Research 32, e40. doi: 10.1093/nar/gnh033.
Beverley, S. M., Owens, K. L., Showalter, M., Griffith, C. L., Doering, T. L., Jones, V. C. and McNeil, M. R. (2005). Eukaryotic UDP-galactopyranose mutase (GLF gene) in microbial and metazoal pathogens. Eukaryotic Cell 4, 11471154. doi: 10.1128/EC.4.6.1147–1154.2005.
Blaxter, M. L., Page, A. P., Rudin, W. and Maizels, R. M. (1992). Nematode surface coats - actively evading immunity. Parasitology Today 8, 243247. doi: 10.1016/0169-4758(92)90126-M.
Briese, M., Esmaeili, B., Johnson, N. M. and Sattelle, D. B. (2006). pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans. Invertebrate Neuroscience 6, 512. doi: 10.1007/s10158-005-0011-x.
Britton, C. and Murray, L. (2002). A cathepsin L protease essential for Caenorhabditis elegans embryogenesis is functionally conserved in parasitic nematodes. Molecular and Biochemical Parasitology 122, 2133. doi: 10.1016/S0166-6851(02)00066-X.
Calixto, A., Chelur, D., Topalidou, I., Chen, X. and Chalfie, M. (2010). Enhanced neuronal RNAi in C. elegans using SID-1. Nature Methods 7, 554559. doi: 10.1038/nmeth.1463.
Correnti, J. M., Brindley, P. J. and Pearce, E. J. (2005). Long-term suppression of cathepsin B levels by RNA interference retards schistosome growth. Molecular and Biochemical Parasitology 143, 209215. doi: 10.1016/j.molbiopara.2005.06.007.
Dalzell, J. J., McVeigh, P., Warnock, N. D., Mitreva, M., Bird, D. M., Abad, P., Fleming, C. C., Day, T. A., Mousley, A., Marks, N. J. and Maule, A. G. (2011). RNAi effector diversity in nematodes. PLoS Neglected Tropical Diseases 5, e1176. doi: 10.1371/journal.pntd.0001176.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. K., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811. doi: 10.1038/35888.
Frand, A. R., Russel, S. and Ruvkun, G. (2005). Functional genomic analysis of C. elegans molting. PLoS Biology 3. doi: 10.1371/journal.pbio.0030312.
Fraser, A. G., Kamath, R. S., Zipperlen, P., Martinez-Campos, M., Sohrmann, M. and Ahringer, J. (2000). Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325330. doi: 10.1038/35042517.
Fritz, J.-A. and Behm, C. A. (2009). CUTI-1: a novel tetraspan protein involved in C. elegans CUTicle formation and epithelial Integrity. PLoS ONE 4, e5117. doi: 10.1371/journal.pone.0005117.
Geldhof, P., Murray, L., Couthier, A., Gilleard, J. S., McLauchlan, G., Knox, D. P. and Britton, C. (2006). Testing the efficacy of RNA interference in Haemonchus contortus. International Journal for Parasitology 36, 801810. doi: 10.1016/j.ijpara.2005.12.004.
Gill, J. H., Redwin, J. M., Van Wyk, J. A. and Lacey, E. (1995). Avermectin inhibition of larval development in Haemonchus contortus – Effects of ivermectin resistance. International Journal for Parasitology 25, 463470. doi: 10.1016/0020-7519(94)00087-5.
Gillan, V., Maitland, K., McCormack, G., Him, N. A. and Devaney, E. (2009). Functional genomics of hsp-90 in parasitic and free-living nematodes. International Journal for Parasitology 39, 10711081. doi: 10.1016/j.ijpara.2009.02.024.
Gilleard, J. S. (2004). The use of Caenorhabditis elegans in parasitic nematode research. Parasitology 128 (Suppl 1), S49S70. doi: 10.1017/S003118200400647X.
Glendinning, S. K., Buckingham, S. D., Sattelle, D. B., Wonnacott, S. and Wolstenholme, A. J. (2011). Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans. PLoS ONE 6, e22390. doi: 10.1371/journal.pone.0022390.
Grant, W. N. and Behm, C. A. (2007). Target identification and validation for anthelmintic discovery. Expert Opinion on Drug Discovery 2, S91S98. doi: 10.1517/17460441.2.S1.S91.
Hetherington, S., Gally, C., Fritz, J.-A., Polanowska, J., Reboul, J., Schwab, Y., Zahreddine, H., Behm, C. and Labouesse, M. (2011). PAT-12, a potential anti-nematode target, is a novel spectraplakin partner essential for C. elegans hemidesmosome integrity and embryonic morphogenesis. Developmental Biology 350, 267278. doi: 10.1016/j.ydbio.2010.11.025.
Hill, A. A., Hunter, C. P., Tsung, B. T., Tucker-Kellogg, G. and Brown, E. L. (2000). Genomic analysis of gene expression in C. elegans. Science 290, 809812. doi: 10.1126/science.290.5492.809.
Issa, Z., Grant, W. N., Stasiuk, S. and Shoemaker, C. B. (2005). Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis. International Journal for Parasitology 35, 935940. doi: 10.1016/j.ijpara.2005.06.001.
Johnson, N. M., Behm, C. A. and Trowell, S. C. (2005). Heritable and inducible gene knockdown in C. elegans using Wormgate and the ORFeome. Gene 359, 2634. doi: 10.1016/j.gene.2005.05.034.
Johnson, N. M., Fritz, J.-A. and Behm, C. A. (2008). RNA interference in Caenorhabditis elegans. In RNA Interference: Methods for Plants and Animals (eds. Doran, T. & Helliwell, C.), pp. 85113. CAB International Wallingford, UK.
Kamath, R. S. and Ahringer, J. (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313321. doi: 10.1016/S1046-2023(03)00050-1.
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P. and Ahringer, J. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231237. doi: 10.1038/nature01278.
Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G. and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biology 2, research0002research0002.0010. doi: 10.1186/gb-2000-2-1-research0002.
Kennedy, S., Wang, D. and Ruvkun, G. (2004). A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature 427, 645649. doi: 10.1038/nature02302.
Kotze, A. C. and Bagnall, N. H. (2006). RNA interference in Haemonchus contortus: suppression of beta-tubulin gene expression in L3, L4 and adult worms in vitro. Molecular and Biochemical Parasitology 145, 101110. doi: 10.1016/j.molbiopara.2005.09.012.
Kotze, A. C., Hines, B. M. and Ruffell, A. P. (in press). A reappraisal of the relative sensitivity of nematode pharyngeal and somatic musculature to macrocyclic lactone drugs. International Journal for Parasitology: Drugs and Drug Resistance. doi: 10.1016/j.ijpddr.2011.10.002.
Kotze, A. C., Le Jambre, L. F. and O'Grady, J. (2006). A modified larval migration assay for detection of resistance to macrocyclic lactones in Haemonchus contortus, and drug screening with Trichostrongylidae parasites. Veterinary Parasitology 137, 294305. doi: 10.1016/j.vetpar.2006.01.017.
Kotze, A. C. and McClure, S. J. (2001). Haemonchus contortus utilises catalase in defence against exogenous hydrogen peroxide in vitro. International Journal for Parasitology 31, 15631571. doi: 10.1016/S0020-7519(01)00303-4.
Krautz-Peterson, G., Radwanska, M., Ndegwa, D., Shoemaker, C. B. and Skelly, P. J. (2007). Optimizing gene suppression in schistosomes using RNA interference. Molecular and Biochemical Parasitology 153, 194202. doi: 10.1016/j.molbiopara.2007.03.006.
Kumar, S., Chaudhary, K., Foster, J. M., Novelli, J. F., Zhang, Y., Wang, S., Spiro, D., Ghedin, E. and Carlow, C. K. S. (2007). Mining predicted essential genes of Brugia malayi for nematode drug targets. PLoS ONE 2, e1189. doi: 10.1371/journal.pone.0001189.
Kwa, M. S., Veenstra, J. G., Van Dijk, M. and Roos, M. H. (1995). β-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans. Journal of Molecular Biology 246, 500510. doi: 10.1006/jmbi.1994.0102.
Lendner, M., Doligalska, M., Lucius, R. and Hartmann, S. (2008). Attempts to establish RNA interference in the parasitic nematode Heligmosomoides polygyrus. Molecular and Biochemical Parasitology 161, 2131. doi: 10.1016/j.molbiopara.2008.06.003.
Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. (2001). Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current Biology 11, 171176. doi: 10.1016/S0960-9822(01)00052-5.
McCarter, J. P. (2004). Genomic filtering: an approach to discovering novel antiparasitics. Trends in Parasitology 20, 462468. doi: 10.1016/j.pt.2004.07.008.
Merritt, C. and Seydoux, G. (2010). Transgenic solutions for the germline. In WormBook (ed. The C. elegans Research Community), doi: 10.1895/wormbook.1.148.1.
Novelli, J. F., Chaudhary, K., Canovas, J., Benner, J. S., Madinger, C. L., Kelly, P., Hodgkin, J. and Carlow, C. K. S. (2009). Characterization of the Caenorhabditis elegans UDP-galactopyranose mutase homolog glf-1 reveals an essential role for galactofuranose metabolism in nematode surface coat synthesis. Developmental Biology 335, 340355. doi: 10.1016/j.ydbio.2009.09.010.
Pfaffl, M. W., Horgan, G. W. and Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Research 30, e36. doi: 10.1093/nar/30.9.e36.
Praitis, V., Casey, E., Collar, D. and Austin, J. (2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 12171226.
Samarasinghe, B., Knox, D. P. and Britton, C. (2011). Factors affecting susceptibility to RNA interference in Haemonchus contortus and in vivo silencing of an H11 aminopeptidase gene. International Journal for Parasitology 41, 5159. doi: 10.1016/j.ijpara.2010.07.005.
Simmer, F., Tijsterman, M., Parrish, S., Koushika, S., Nonet, M., Fire, A., Ahringer, J. and Plasterk, R. (2002). Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Current Biology 12, 1317. doi: 10.1016/S0960-9822(02)01041-2.
Sönnichsen, B., Koski, L. B., Walsh, A., Marschall, P., Neumann, B., Brehm, M., Alleaume, A. M., Artelt, J., Bettencourt, P., Cassin, E., Hewitson, M., Holz, C., Khan, M., Lazik, S., Martin, C., Nitzsche, B., Ruer, M., Stamford, J., Winzi, M., Heinkel, R., Roder, M., Finell, J., Hantsch, H., Jones, S. J., Jones, M., Piano, F., Gunsalus, K. C., Oegema, K., Gonczy, P., Coulson, A., Hyman, A. A. and Echeverri, C. J. (2005). Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans. Nature 434, 462469. doi: 10.1038/nature03353.
Tabara, H., Grishok, A. and Mello, C. C. (1998). RNAi in C. elegans: soaking in the genome sequence. Science 282, 430431. doi: 10.1126/science.282.5388.430.
Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. and Driscoll, M. (2000). Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nature Genetics 24, 180183. doi: 10.1038/72850.
Timmons, L. (2006). Delivery methods for RNA interference in C. elegans. In C. elegans: Methods and Applications (ed. Strange, K.), pp. 119125. Humana Press Inc, Totowa, NJ.
Timmons, L., Court, D. L. and Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103112. doi: 10.1016/S0378-1119(00)00579-5.
Timmons, L. and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 894. doi: 10.1038/27579.
Visser, A., Geldhof, P., de Maere, V., Knox, D. P., Vercruysse, J. and Claerebout, E. (2006). Efficacy and specificity of RNA interference in larval life-stages of Ostertagia ostertagi. Parasitology 133, 777783. doi: 10.1017/S0031182006001004.
Wang, D., Kennedy, S., Conte, D. Jr., Kim, J. K., Gabel, H. W., Kamath, R. S., Mello, C. C. and Ruvkun, G. (2005). Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants. Nature 436, 593597. doi: 10.1038/nature04010.
Xu, J., Cheng, T., Feng, H. T., Pavlos, N. J. and Zheng, M. H. (2007). Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis. Histology and Histopathology 22, 443454.

Keywords

Silencing of essential genes by RNA interference in Haemonchus contortus

  • J. L. ZAWADZKI (a1), A. C. KOTZE (a2), J.-A. FRITZ (a3), N. M. JOHNSON (a3), J. E. HEMSWORTH (a1), B. M. HINES (a2) and C. A. BEHM (a3)...

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