Degree Type

Dissertation

Date of Award

2012

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

First Advisor

Richard J. Martin

Second Advisor

Alan P. Robertson

Abstract

Parasitic nematode infections of humans, plants and animals are of major economic impact. These parasites cause losses of billions of dollars per year in crop damage and through livestock infection; the infections to humans are equally debilitating. Anthelmintics are the main chemotherapeutic agents used for treatment and prophylaxis of nematode infections because there is presently no effective vaccine on the market. Most of the anthelmintics presently used in treating nematode infections in humans were first developed for use in animals. In most cases, these anthelmintics have been used in humans without changing the properties of the drugs at all. However, resistance has been reported to the mainstay anthelmintics, namely AChR agonists (levamisole, pyrantel), benzimidazoles (albendazole) and macrocyclic lactones (ivermectin). There is therefore the urgent need to understand the genetics of the receptors targeted by these anthelmintics and the mechanisms of resistance, with the view to improving efficacy of the presently used drugs. In addition, there is the need to find alternative targets for developing new anthelmintics as well as fully studying the mechanism of action of any new anthelmintic.

We have demonstrated the effects of the new novel-acting cyclooctadepsipeptide anthelmintic, emodepside, on the membrane potential and voltage-activated currents in the pig parasite Ascaris suum. Emodepside hyperpolarized the membrane in a slow, time-dependent manner without changing the input conductance. We show that the purported emodepside target receptors in C. elegans, SLO-1 and LAT-1 are expressed in the muscle flap of A. suum (Asu-slo-1 and Asu-lat-1). Emodepside potentiated the voltage-activated Ca2+-dependent K+ channel currents in a time- and voltage-dependent manner. We have demonstrated that emodepside effect on the K+ channel currents is inhibited by iberiotoxin, a selective SLO-1 channel inhibitor. The effects of emodepside on the membrane potential and K+ channel currents were modulated by NO and protein kinase activators/inhibitors. Last but not least, we demonstrate that diethylcarbamazine (DEC), a filarial anthelmintic, potentiates the effects of emodepside on the membrane potential and SLO-1-like currents. Our results clearly demonstrate effects of emodepside in a parasitic nematode and the modulation of emodepside effects by second messengers like protein kinases. We also show that a formulation of emodepside and DEC could be used for treating filarial parasites, slowing the development of resistance to emodepside.

Finally, we show the cloning of four acetylcholine receptor subunit genes from another pig parasite, Oesophagostomum dentatum and the expression and characterization of these receptor subunits in Xenopus laevis oocytes. By employing the three ancillary factors of Haemonchus contortus, Hc-ric-3.1, Hc-unc-50 and Hc-unc-74, we have characterized four levamisole receptor subtypes of O. dentatum with different pharmacological properties. First, the receptor subtype we have termed Pyr-nAChR, was composed of Ode-unc-29 and Ode-unc-63 and responded to pyrantel as the most potent agonist. The second receptor subtype, Pyr/Tbd-nAChR, responded to pyrantel and tribendimidine as the most potent agonists and was composed of Ode-unc-29, Ode-unc-63 and Ode-unc-38. The third receptor subtype, nAChR, responded to ACh as the most potent agonist and was composed of Ode-unc-29, Ode-unc-63 and Ode-acr-8. The last receptor subtype, Lev-nAChR, responded to levamisole as the most potent agonist and was composed of Ode-unc-29, Ode-unc-63, Ode-acr-8 and Ode-unc-38. In the Lev-nAChR, derquantel distinguished receptor subtypes with pA2 values of 6.8 and 8.4 for levamisole and pyrantel, respectively. The calcium permeability (PCa/PNa) of three receptor subtypes differed. We measured PCa/PNa of 10.3, 0.38 and 0.38 for the Lev-nAChR, Pyr/Tbd-nAChR and nAChR subtypes, respectively. Unlike the receptors in Caenorhabditis elegans and Haemonchus contortus, all three ancillary factors were not absolutely required to reconstitute O. dentatum functional levamisole receptors. We reconstituted receptors with robust responses to the agonists when we sequentially removed these ancillary factors. However, removal of all three factors did not reconstitute any receptors, demonstrating the need for at least one of these ancillary factors. Our results demonstrate the plasticity in the levamisole receptors of O. dentatum and suggest that the subtypes may have different physiological roles and/or expressed in different tissues of the parasite.

Copyright Owner

Samuel Buxton

Language

en

File Format

application/pdf

File Size

255 pages

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