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Title: Characterisation of ACR-26 - a novel acetylcholine receptor of parasitic nematodes
Author: Bennett, Hayley Marie
ISNI:       0000 0004 2745 2963
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2011
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Parasititic nematodes are important aetiological agents of disease, infecting humans and livestock worldwide. Nicotinic acetylcholine receptors (nAChRs) in the nematode neuromuscular system are proven anthelmintic targets. This project aimed to characterise a novel nAChR subunit gene, acr-26, found in A. suum, assessing its potential as a drug target. Using both bioinformatic and experimental approaches, evidence of acr-26 was found in multiple parasitic nematode species of clinical and economical importance, conserved over three distinct evolutionary clades. However, no evidence of an acr-26 sequence was found in any free-living or plant parasitic nematode, suggesting that this subunit is more specific to vertebrate parasitic nematodes, which would make it a good drug target. Alongside acr-26, nine orthologues of C. elegans nAChR subunits were identified in a recent A. suum transcriptome dataset; acr-S, acr-l S, acr-16, acr-I Z, acr-21, deg-3, unc-29, unc-38 and unc-63. This diversity was on par with another clade III parasite, B. malayi. Acr-26 appeared to constitute a distinct subunit family. The biological localisation of the nAChR subunit mRNAs was investigated with RT-PCR in dissected adult A. suum - acr-26 was ubiquitously indicated. Interestingly, exclusive expression in isolated pharyngeal tissue indicates acr-21 as a candidate drug target. Immunocytochemistry with an antibody directed against the A. suum ACR-26 subunit showed expression in head muscle cells but not in mid-body wall muscle cells from the adult nematode; it is therefore likely that ACR-26 forms a nAChR subtype not previously characterised in vivo. Two-electrode voltage clamp electrophysiology revealed exogenously expressed homomeric ACR-26 receptors in Xenopus oocytes to be sensitive to both acetylcholine (ECso = 42 nM, 95 % confidence interval = 15 nM to 109 nM) and nicotine (ECso = 25 u M, 95 % confidence interval = 15 ~M to 42 ~M,). Initial results also suggested a lack of sensitivity to the anthelmintic levamisole. Unpredictable receptor expression necessitated an alternative approach for functional evaluation. An inducible stable cell line was developed, however functional activity was not forthcoming. The challenges of heterologous nAChR expression led to investigation AChR chaperone protein RIC-3 and its host specificity. In conclusion, this study has found a species distribution that indicates ACR-26 as a promising drug target. The expression pattern in vivo and the in vitro pharmacology of an ACR-26 receptor support its consideration as a unique, and hence potentially anthelmintic resistance-busting, nAChR target.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available