Investigation of Acetylcholine Receptor Diversity in a Nematode Parasite Leads to Characterization of Tribendimidine- and Derquantel-Sensitive nAChRs


Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.


Vyšlo v časopise: Investigation of Acetylcholine Receptor Diversity in a Nematode Parasite Leads to Characterization of Tribendimidine- and Derquantel-Sensitive nAChRs. PLoS Pathog 10(1): e32767. doi:10.1371/journal.ppat.1003870
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003870

Souhrn

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.


Zdroje

1. HotezPJ, BrindleyPJ, BethonyJM, KingCH, PearceEJ, et al. (2008) Helminth infections: the great neglected tropical diseases. The Journal of Clinical Investigation 118: 1311–1321.

2. HotezPJ, MolyneuxDH, FenwickA, KumaresanJ, SachsSE, et al. (2007) Control of Neglected Tropical Diseases. The New England Journal of Medicine 357: 1018–1027.

3. BrownLA, JonesAK, BuckinghamSD, MeeCJ, SattelleDB (2006) Contributions from Caenorhabditis elegans functional genetics to antiparasitic drug target identification and validation: Nicotinic acetylcholine receptors, a case study. International Journal for Parasitology 36: 617–624.

4. WolstenholmeAJ, FairweatherI, PrichardR, von Samson-HimmelstjernaG, SangsterNC (2004) Drug resistance in veterinary helminths. Trends in Parasitology 20 (10) 469–476.

5. MartinRJ, RobertsonAP (2007) Mode of action of Levamisole and pyrantel, anthelmintic resistance, E153 and Q57. Parasitology 134: 1093–1104.

6. XiaoS, Hui-MingW, TannerM, UtzingerJ, ChongW (2005) Tribendimidine: a promising, safe and broad-spectrum anthelmintic agent from China. Acta Trop 94 (1) 1–14.

7. KaminskyR, GauvryN, Schorderet WeberS, SkripskyT, BouvierJ, et al. (2008) Identification of the amino-acetonitrile derivative monepantel (AAD 1566) as a new anthelmintic drug development candidate. Parasitol Res 103 (4) 931–939.

8. KaminskyR, DucrayP, JungM, CloverR, RufenerL, et al. (2008) A new class of anthelmintics effective against drug-resistant nematodes. Nature 452: 176–180.

9. RobertsonAP, ClarkCL, BurnsTA, ThompsonDP, GearyTG, et al. (2002) Paraherquamide and 2-Deoxy-paraherquamide Distinguish Cholinergic Receptor Subtypes in Ascaris suum. The Journal of Pharmacology & Experimental Therapeutics 302: 853–860.

10. XiaoS-H, UtzingerJ, TannerM, KeiserJ, XueJ (2013) Advances with the Chinese anthelmintic drug tribendimidine in clinical trials and laboratory investigations. Acta Trop 126 (2) 115–126.

11. HuY, XiaoS-H, AroianRV (2009) The new anthelmintic tribendimidine is an L-type (Levamisole and Pyrantel) nicotinic acetylcholine receptor agonist. PLoS Negl Trop Dis 3 (8) e499.

12. LeeB, ClothierM, DuttonF, NelsonS, JohnsonS, et al. (2002) Marcfortine and paraherquamide class of anthelmintics: discovery of PNU-141962. Curr Top Med Chem 2 (7) 779–793.

13. QianH, MartinRJ, RobertsonAP (2006) Pharmacology of N-, L-, and B-subtypes of nematode nAChR resolved at the single-channel level in Ascaris suum. The FASEB Journal 20: E2108–E2116.

14. WilliamsonSM, RobertsonAP, BrownL, WilliamsT, WoodsDJ, et al. (2009) The Nicotinic Acetylcholine Receptors of the Parasitic Nematode Ascaris suum: Formation of Two Distinct Drug Targets by Varying the Relative Expression Levels of Two Subunits. PloS PATHOGENS 5: e1000517.

15. NeveuC, CharvetCL, FauvinA, CortetJ, BeechRN, et al. (2010) Genetic diversity of levamisole receptor subunits in parasitic nematode species and abbreviated transcripts associated with resistance. Pharmacogenetics and Genomics 20: 414–425.

16. BoulinT, FauvinA, CharvetCL, CortetJ, CabaretJ, et al. (2011) Functional reconstitution of Haemonchus contortus acetylcholine receptors in Xenopus oocytes provides mechanistic insights into levamisole resistance. Br J Pharmacol 164: 1421–1432.

17. MartinRJ (1997) Modes of action of anthelmintic drugs. Veterinary Journal 154: 11–34.

18. RichmondJE, JorgensenEM (1999) One GABA and two acetylcholine receptors function at the C. elegans neuromuscular junction. Nature Neuroscience 2: 791–797.

19. CulettoE, BaylisHA, RichmondJE, JonesAK, FlemingJT, et al. (2004) The Caenorhabditis elegans unc-63 Gene Encodes a Levamisole-sensitive Nicotinic Acetylcholine Receptor alpha Subunit. The Journal of Biological Chemistry 279: 42476–42483.

20. FlemingJT, SquireMD, BarnesTM, TornoeC, MatsudaK, et al. (1997) Caenorhabditis elegans levamisole resistance genes lev-1, unc-29 and unc-38 encode functional nicotinic acetylcholine receptor subunits. J Neurosci 17: 5843–5857.

21. TowersPR, EdwardsB, RichmondJE, SattelleDB (2005) The Caenorhabditis elegans lev-8 gene encodes a novel type of nicotinic acetylcholine receptor alpha subunit. J Neurochem 93: 1–9.

22. LewisJA, WuC-H, BergH, LevineJH (1980) The genetics of levamisole resistance in the nematode Caenorhabditis elegans. Neuroscience 5: 967–989.

23. TouroutineD, FoxR, Von StetinaS, AB, MillerDr, et al. (2005) ACR-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the Caenorhabditis elegans neuromuscular junction. J Biol Chem 280 (29) 27013–27021.

24. RobertsonAP, BjornHE, MartinRJ (1999) Resistance to levamisole resolved at the single-channel level. The FASEB Journal 13: 749–760.

25. RobertsonAP, BjornHE, MartinRJ (2000) Pyrantel resistance alters nematode nicotinic acetylcholine receptor single-channel properties. European Journal of Pharmacology 394: 1–8.

26. BoulinT, GielenM, RichmondJE, WilliamsDC, PaolettiP, et al. (2008) Eight genes are required for functional reconstitution of the Caenorhabditis elegans levamisole-sensitive acetylcholine receptor. PNAS 105: 18590–18595.

27. QianH, RobertsonAP, Powell-CoffmanJ, MartinRJ (2008) Levamisole resistance resolved at the single-channel level in Caenorhabditis elegans. FASEB J 22: 3247–3254.

28. JonesAK, DavisP, HodgkinJ, SattelleDB (2007) The nicotinic acetylcholine receptor gene family of the nematode Caenorhabditis elegans: an update on nomenclature. Invert Neurosci 7 (2) 129–131.

29. BlaxterML, De LeyP, GareyJ, LiuL, ScheldemanP, et al. (1998) A molecular evolutionary framework for the phylum Nematoda. Nature 392 (6671) 71–75.

30. RobertsonSJ, PenningtonAJ, EvansAM, MartinRJ (1994) The action of pyrantel as an agonist and an open channel blocker at acetylcholine receptors in isolated Ascaris suum muscle vesicles. Eur J Pharmacol 271 (2–3) 273–282.

31. ChangY, HuangY, WhiteakerP (2010) Mechanism of allosteric modulation of the cys-loop receptors. Pharmaceuticals 3: 2592–2609.

32. VerninoS, AmadorM, LuetjeCW, PatrickJ, DaniJA (1992) Calcium modulation and high calcium permeability of neuronal nicotinic acetylcholine receptors. Neuron 8: 127–134.

33. KeiserJ, TrittenL, AdelfioR, vargasM (2012) Effect of combinations of marketed human anthelmintic drugs against Trichuris muris in vitro and in vivo. Parasites & Vectors 5: 292.

34. AlmedomRB, LiewaldJF, HernandoG, SchultheisC, RayesD, et al. (2009) An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse. EMBO J 28: 2636–2649.

35. EimerS, GottschalkA, HengartnerM, HorvitzHR, RichmondJ, et al. (2007) Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50. EMBO J 26: 4313–4323.

36. GottschalkA, AlmedomRB, SchedletzkyT, AndersonSD, YatesJRIII, et al. (2005) Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans. EMBO J 24: 2566–2578.

37. HaleviS, McKayJ, PalfreymanM, YassinL, EshelM, et al. (2002) The C. elegans ric-3 gene is required for maturation of nicotinic acetylcholine receptors. EMBO J 21: 1012–1020.

38. HaugstetterJ, BlicherT, EllgaardL (2005) Identification and characterization of a novel thioredoxin-related transmembrane protein of the endoplasmic reticulum. J Biol Chem 280: 8371–8380.

39. TouroutineD, FoxRM, Von StetinaSE, BurdinaA, MillerDMIII, et al. (2005) ACR-16 encodes an essential subunit of the levamisole-resistant nicotinic receptor at the C. elegans neuromuscular junction. J Biol Chem 280 (29) 27013–27021.

40. VaradyM, BjornH, CravenJ, NansenP (1997) In vitro characterization of lines of Oesophagostomum dentatum selected or not selected for resistance to pyrantel, levamisole and ivermectin. International Journal for Parasitology 27: 77–81.

41. KatohK, MisawaK, KumaK, MiyataT (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30: 3059–3066.

42. GuindonS, GascuelO (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704.

43. BrownLA, JohnsonBA, GoodmanMB (2008) Patch clamp recording of ion channels expressed in Xenopus oocytes. J Vis Exp 20: e936 910.3791/3936.

44. EdgarRC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.

45. ImotoK, BuschC, SakmannB, MishinaM, KonnoT, et al. (1988) Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335 (13) 645–648.

46. ImotoK, MethfesselC, SakmannB, MishinaM, MoriY, et al. (1986) Location of a δ-subunit region determining ion transport through the acetylcholine receptor channel. Nature 324 (6098) 670–674.

47. EdgarR (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.

48. SaitouN, NeiM (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406–425.

49. Ben-AmiHC, BialaY, FarahH, ElishevitzE, BattatE, et al. (2009) Receptor and subunit specific interactions of RIC-3 with Nicotinic Acetylcholine Receptors. Biochemistry 48: 12329–12336.

50. MillarNS (2008) RIC-3: a nicotinic acetylcholine receptor chaperone. British Journal of Pharmacology 153: S177–S183.

51. LansdellSJ, GeeVJ, HarknessPC, DowardAI, BakerER, et al. (2005) RIC-3 enhances functional expressioin of multiple nicotinic acetylcholine receptor subtypes in mammalian cells. Mol Pharmacol 68: 1431–1438.

52. Ben-AmiHC, YassinL, FarahH, MichaeliA, EshelM, et al. (2005) RIC-3 affects properties and quantity of nicotinic acetylcholine receptors via a mechanism that does not require the coiled-coil domains. The Journal of Biological Chemistry 280: 28053–28060.

53. EimerS, GottschalkA, HengartnerM, HorvitzHR, RichmondJ, et al. (2007) Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50. EMBO J 26: 4313–4323.

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Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

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