Hostname: page-component-745bb68f8f-b6zl4 Total loading time: 0 Render date: 2025-01-13T12:39:48.641Z Has data issue: false hasContentIssue false
  • English
  • Français

P-glycoproteins of Haemonchus contortus: development of real-time PCR assays for gene expression studies

Published online by Cambridge University Press:  01 June 2011

S.M. Williamson  and
A.J. Wolstenholme
S.M. Williamson
Affiliation:
Department of Infectious Diseases, College of Veterinary Medicine and Center for Tropical and Emerging Global Disease, University of Georgia, Athens, GA30602, USA
A.J. Wolstenholme*
Affiliation:
Department of Infectious Diseases, College of Veterinary Medicine and Center for Tropical and Emerging Global Disease, University of Georgia, Athens, GA30602, USA
*
* E-mail: adrianw@uga.edu
Get access Rights & Permissions [Opens in a new window]

Abstract

P-glycoproteins (P-gps) are proteins that function as efflux pumps, removing lipophilic xenobiotic compounds from cells. There is evidence that P-gps play a role in the resistance of parasitic nematodes to anthelmintic drugs such as benzimidazoles and macrocyclic lactones. As anthelmintic resistance becomes more common, it is important to identify candidate resistance genes with the aim of understanding the molecular basis of resistance, and of developing assays to detect these resistance-associated changes. We identified several sequences from the genome of the parasite Haemonchus contortus with convincing homology to the known P-gp coding genes of the model nematode Caenorhabditis elegans. Nine of these sequences were successfully amplified by polymerase chain reaction (PCR) and shown to be most similar to the C. elegans sequences for pgp-1, pgp-2, pgp-3, pgp-4, pgp-9, pgp-10, pgp-11, pgp-12 and pgp-14. These partial P-gp sequences from H. contortus were used to design and optimize a quantitative real-time PCR assay to investigate potential changes in the expression levels of P-gp transcripts associated with drug resistance. No significant changes in P-gp mRNA expression levels were found in a rapidly selected ivermectin-resistant parasite isolate compared to its drug-sensitive parent, but the assay has the potential to be used on other isolates in the future to further investigate resistance-associated changes in P-gp gene expression.

Type
Research Papers
Information
Journal of Helminthology , Volume 86 , Issue 2 , June 2012 , pp. 202 - 208
Copyright
Copyright © Cambridge University Press 2011

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ardelli, B.F., Stitt, L.E. & Tompkins, J.B. (2010) Inventory and analysis of ATP-binding cassette (ABC) systems in Brugia malayi. Parasitology 137, 1195–1212.CrossRefGoogle ScholarPubMed
Barnes, E.H., Dobson, R.J., Stein, P.A., Le Jambre, L.F. & Lenane, I.J. (2001) Selection of different genotype larvae and adult worms for anthelmintic resistance by persistent and short-acting avermectin/milbemycins. International Journal for Parasitology 31, 720–727.CrossRefGoogle ScholarPubMed
Bartley, D.J., McAllister, H., Bartley, Y., Dupuy, J., Menez, C., Alvinerie, M., Jackson, F. & Lespine, A. (2009) P-glycoprotein interfering agents potentiate ivermectin susceptibility in ivermectin sensitive and resistant isolates of Teladorsagia circumcincta and Haemonchus contortus. Parasitology 136, 1081–1088.Google Scholar
Beech, R.N., Wolstenholme, A.J., Neveu, C. & Dent, J.A. (2010) Naming genes in the parasitic nematodes. Trends in Parasitology 26, 334–340.CrossRefGoogle Scholar
Blackhall, W.J., Liu, H.Y., Xu, M., Prichard, R.K. & Beech, R.N. (1998) Selection at a P-glycoprotein gene in ivermectin- and moxidectin-selected strains of Haemonchus contortus. Molecular and Biochemical Parasitology 95, 193–201.CrossRefGoogle Scholar
Blackhall, W.J., Prichard, R.K. & Beech, R.N. (2008) P-glycoprotein selection in strains of Haemonchus contortus resistant to benzimidazoles. Veterinary Parasitology 152, 101–107.CrossRefGoogle ScholarPubMed
Bourguinat, C., Ardelli, B.F., Pion, S.D., Kamgno, J., Gardon, J., Duke, B.O., Boussinesq, M. & Prichard, R.K. (2008) P-glycoprotein-like protein, a possible genetic marker for ivermectin resistance selection in Onchocerca volvulus. Molecular and Biochemical Parasitology 158, 101–111.CrossRefGoogle ScholarPubMed
Coles, G.C., Rhodes, A.C. & Wolstenholme, A.J. (2005) Rapid selection for ivermectin resistance in Haemonchus contortus. Veterinary Parasitology 129, 345–347.Google Scholar
Fauvin, A., Charvet, C., Issouf, M., Cortet, J., Cabaret, J. & Neveu, C. (2010) cDNA-AFLP analysis in levamisole-resistant Haemonchus contortus reveals alternative splicing in a nicotinic acetylcholine receptor subunit. Molecular and Biochemical Parasitology 170, 105–107.CrossRefGoogle Scholar
Hodgkinson, J.E., Clark, H.J., Kaplan, R.M., Lake, S.L. & Matthews, J.B. (2008) The role of polymorphisms at β-tubulin isotype 1 codons 167 and 200 in benzimidazole resistance in cyathostomins. International Journal for Parasitology 38, 1149–1160.CrossRefGoogle ScholarPubMed
Howell, S.B., Burke, J.M., Miller, J.E., Terrill, T.H., Valencia, E., Williams, M.J., Williamson, L.H., Zajac, A.M. & Kaplan, R.M. (2008) Prevalence of anthelmintic resistance on sheep and goat farms in the southeastern United States. Journal of the American Veterinary Medicine Association 233, 1913–1919.CrossRefGoogle ScholarPubMed
Hunt, P.W., Knox, M.R., Le Jambre, L.F., McNally, J. & Anderson, L.J. (2008) Genetic and phenotypic differences between isolates of Haemonchus contortus in Australia. International Journal for Parasitology 38, 885–900.CrossRefGoogle ScholarPubMed
James, C.E. & Davey, M.W. (2009) Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans. International Journal for Parasitology 39, 213–220.CrossRefGoogle ScholarPubMed
Kamath, R.S. & Ahringer, J. (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313–321.Google Scholar
Kerboeuf, D., Blackhall, W., Kaminsky, R. & von Samson-Himmelstjerna, G. (2003a) P-glycoprotein in helminths: function and perspectives for anthelmintic treatment and reversal of resistance. International Journal of Antimicrobial Agents 22, 332–346.CrossRefGoogle ScholarPubMed
Kerboeuf, D., Guegnard, F. & Le Vern, Y. (2003b) Detection of P-glycoprotein-mediated multidrug resistance against anthelmintics in Haemonchus contortus using anti-human mdr1 monoclonal antibodies. Parasitology Research 91, 79–85.CrossRefGoogle ScholarPubMed
Kotze, A.C. & Bagnall, N.H. (2006) RNA interference in Haemonchus contortus: suppression of β-tubulin gene expression in L3, L4 and adult worms in vitro. Molecular and Biochemical Parasitology 145, 101–110.CrossRefGoogle ScholarPubMed
Maeda, I., Kohara, Y., Yamamoto, M. & Sugimoto, A. (2001) Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current Biology 11, 171–176.Google Scholar
Molin, E.U. & Mattsson, J.G. (2008) Effect of acaricides on the activity of glutathione transferases from the parasitic mite Sarcoptes scabiei. Parasitology 135, 115–123.Google Scholar
Otsen, M., Plas, M.E., Lenstra, J.A., Roos, M.H. & Hoekstra, R. (2000) Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus. Molecular and Biochemical Parasitology 110, 69–77.Google Scholar
Ouellette, M. & Borst, P. (1991) Drug resistance and P-glycoprotein gene amplification in the protozoan parasite Leishmania. Research in Microbiology 142, 737–746.CrossRefGoogle ScholarPubMed
Peter, J.W. & Chandrawathani, P. (2005) Haemonchus contortus: parasite problem No. 1 from tropics – Polar Circle. Problems and prospects for control based on epidemiology. Tropical Biomedicine 22, 131–137.Google ScholarPubMed
Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29, e45.Google Scholar
Prichard, R.K. & Roulet, A. (2007) ABC transporters and β-tubulin in macrocyclic lactone resistance: prospects for marker development. Parasitology 134, 1123–1132.Google Scholar
Riou, M., Guegnard, F., Sizaret, P.Y., Le Vern, Y. & Kerboeuf, D. (2010) Drug resistance is affected by colocalization of P-glycoproteins in raft-like structures unexpected in eggshells of the nematode Haemonchus contortus. Biochemistry and Cell Biology-Biochimie et Biologie Cellulaire 88, 459–467.Google Scholar
Roos, M.H., Otsen, M., Hoekstra, R., Veenstra, J.G. & Lenstra, J.A. (2004) Genetic analysis of inbreeding of two strains of the parasitic nematode Haemonchus contortus. International Journal for Parasitology 34, 109–115.CrossRefGoogle ScholarPubMed
von Samson-Himmelstjerna, G., Walsh, T.K., Donnan, A.A., Carriere, S., Jackson, F., Skuce, P.J., Rohn, K. & Wolstenholme, A.J. (2009) Molecular detection of benzimidazole resistance in Haemonchus contortus as a tool for routine field diagnosis. Parasitology 136, 349–358.CrossRefGoogle Scholar
Wolstenholme, A.J., Fairweather, I., Prichard, R., von Samson-Himmelstjerna, G. & Sangster, N.C. (2004) Drug resistance in veterinary helminths. Trends in Parasitology 20, 469–476.Google Scholar
Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R. & Prichard, R. (1998) Ivermectin resistance in nematodes may be caused by alteration of P-glycoprotein homolog. Molecular and Biochemical Parasitology 91, 327–335.CrossRefGoogle ScholarPubMed
Zhao, Z., Sheps, J.A., Ling, V., Fang, L.L. & Baillie, D.L. (2004) Expression analysis of ABC transporters reveals differential functions of tandemly duplicated genes in Caenorhabditis elegans. Journal of Molecular Biology 344, 409–417.Google Scholar