Degree Type

Dissertation

Date of Award

2021

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

Major

Genetics and Genomics

First Advisor

Richard J. Martin

Abstract

The prevalence of parasitic nematode infections are a major human and animal health concern. There are still no effective vaccines available, hence anthelmintic drugs have remained the cornerstone for prophylaxis and treatment. The repertoire of available anthelmintics is limited, with treatment relying heavily on three major chemical classes of anthelmintics. These are the imidazothiazoles/tetrahydropyrimidines (levamisole, pyrantel, morantel, oxantel); benzimidazoles (mebendazole, flubendazole, thiabendazole, albendazole); and the macrocyclic lactones (ivermectin, moxidectin, abamectin), all of which act on parasitic nematode ion channels, except for the benzimidazoles. Ion-channels are crucial components of excitable tissues and valuable targets for anthelmintics. Prolong treatment and incorrect use of these anthelmintics however, have led to the development of resistance worldwide. Additionally, the development of new anthelmintics is slow-paced, with only three drug classes being developed and approved for animal use in the since the year 2000. This includes the amino-acetonitrile (monepantel), cyclooctadepsipeptide (emodepside) and the spiroindole (derquantel). Hence, there is an urgent need for the development of new, more effective anthelmintic drugs that can alleviate the morbidity and mortality caused by existing parasite infections. Additionally, significant gaps in our understanding of anthelmintic resistance need to be improved so that we can provide practical solutions on improving drug efficacy and delaying the onset of resistance. We have confirmed the expression of four nicotinic acetylcholine receptor (nAChR) subunits: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8 that constitute the putative levamisole receptor in adult female Ascaris suum intestine. We then validated these findings by using RNAscope in situ hybridization to localize the subcellular distribution of the subunits in the intestine. Quantitative real-time PCR (qPCR) was also used to confirm the mRNA expression levels of each subunit in both muscle and intestine cells. To determine whether these subunits formed functional receptors that were responsive to cholinergic agonists, we employed calcium imaging. Our calcium imaging results demonstrated that both acetylcholine and levamisole elicited intracellular calcium responses in the intestinal tissue. These findings suggest that the presence of functional nAChRs in the intestine may not be limited to neuromuscular transmission, but an acetylcholine paracrine function. Hence, A. suum intestine can be a suitable target for therapeutic exploitation. Secondly, we expressed two receptor subtypes, namely Ode-levamisole and Ode pyrantel/tribendimidine from the pig parasite Oesophagostomum dentatum in Xenopus laevis oocytes. We demonstrated that compounds from the two macrocyclic lactone sub-family, the avermectins (abamectin and ivermectin) and the milbemycin, moxidectin are positive allosteric modulators (PAMs) on the Ode levamisole receptor. In contrast, abamectin and ivermectin acted as negative allosteric modulators (NAMs) on the Ode pyrantel/tribendimidine receptor subtype, while moxidectin maintained its PAM action. These findings suggest that the macrocyclic lactones are allosteric modulators of nAChRs and structural differences between each drug or the presence or absence of a subunit, namely ACR-8 may influence the allosteric modulatory effects. Hence combination therapy that includes macrocyclic lactones and cholinergic anthelmintics, might improve drug efficacy and delay anthelmintic resistance. Finally, we investigated the adaptation of Brugia malayi to levamisole exposure. We showed that B. malayi recovered motility with loss of sensitivity to levamisole within 4 hours of exposure. Molecular analysis also revealed up-regulation of mRNA levels for one AChR subunit, unc-38 and down-regulation of a gene that encodes for an ER retention protein, nra-2. Patch-clamp experiments on 4 hour recovered worms also showed that muscle responses to levamisole had desensitized. Knock down of nra-2 by RNAi resulted in faster recovery in motility, significant reduction in levamisole currents and no change in acetylcholine currents. This suggest that loss of NRA-2 facilitates the insertion of pentameric AChR subtypes in the muscle that are insensitive to levamisole, thus leading to faster recovery in motility in the presence of levamisole. Additionally, simultaneous knockdown of AChR subunits, namely, unc-38, acr-26 and acr-16, inhibited recovery of motility in the worms. These findings are notable and highlights the dynamic mechanisms used to by the parasite to vary AChR subunit composition that generates various receptor subtypes, thus facilitating recovery of motility and insensitivity to anthelmintic exposure (levamisole). This process of habituation can be interpreted as a mechanism of resistance that can be used by parasitic nematodes.

DOI

https://doi.org/10.31274/etd-20210609-117

Copyright Owner

Mark Andrew McHugh

Language

en

File Format

application/pdf

File Size

205 pages

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