Anthelmintic resistance and homeostatic plasticity (Brugia malayi)

Kashyap, Sudhanva S.; Verma, Saurabh; McHugh, Mark; Wolday, Mengisteab; Williams, Paul D.; Robertson, Alan P.; Martin, Richard J.

doi:10.1038/s41598-021-93911-4

Cited by 9 publications

(17 citation statements)

References 60 publications

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“…In addition, the relative order of response to a panel of agonists was the same for each species. This is consistent with the fact that a response to nicotine in living B. malayi is found and its characteristics are unremarkable [29,42]. An alternative to pharmacology changes could be that fewer receptors were expressed at the surface of oocytes ex vivo.…”

Section: Discussionsupporting

confidence: 83%

“…No meaningful difference in the way the N-AChR from different species responded to various agonists, coupled with delayed maximal responses in more derived receptors suggested that a decrease in overall receptor function was likely responsible for the decreased response. The nicotine response of B. malayi in vivo [29,42], the failure to reconstitute a receptor ex vivo , together with a delayed time to maximal response for the species closest to B. malayi suggests a change that affected expression ex vivo specifically. This could be due to an increased dependence one or more members of the network of regulatory accessory proteins that were not included in the cRNA injected into oocytes.…”

Section: Resultsmentioning

confidence: 99%

“…NRA-2 is a nicalin-like homolog and NRA-4 is a NOMO-like homolog [23,40]. In B. malayi , NRA-2 is thought to sense intracellular influx of calcium and regulate the levels of the different L-AChR subunits to produce receptors with different subunit combinations [42]. In C. elegans , NRA-2 and NRA-4 KO worms had reduced L-and N-AChR responses and the L-AChR had altered properties [40].…”

Section: Discussionmentioning

confidence: 99%

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Reconstitution of an N-AChR from Brugia malayi

Noonan

Beech

2022

Preprint

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Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal current to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to maximal response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

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Section: Discussionsupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Reconstitution of an N-AChR from Brugia malayi

Noonan

Beech

2022

Preprint

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“…These include the clade IIIb nematode, A. suum that encodes an N-AChR substantially similar to that found in the clade V nematode C. elegans [1,24,36]. The genome of the clade IIIc filarial parasite, B. malayi [41], also contains an acr-16 that is expected to encode a functional N-AChR since the worm responds to nicotine (NIC) in vivo with an influx of cations as expected from functional AChRs [29,42]. Perhaps surprisingly, the B. malayi N-AChR failed to reconstitute a functional homomeric receptor ex vivo in the presence of RIC-3.…”

Section: Acr-16 Is Conserved Across Nematodesmentioning

confidence: 94%

Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites

Noonan

Beech

2022

PLoS Pathog

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Neurotransmission is an important target for anthelmintic drugs, where receptor characteristics and response can be examined through reconstitution ex vivo in Xenopus laevis oocytes. The homomeric ACR-16 nicotine sensitive acetylcholine receptors (N-AChRs) of several helminth species have been characterized in this way. Our efforts to reconstitute the N-AChR from the clade III filarial parasite, Brugia malayi using similar conditions, initially produced no detectable response. A robust response to acetylcholine is obtained from the closely related clade III parasite Ascaris suum, suggesting that specific changes have occurred between Ascaris and Brugia. N-AChRs from three species intermediate between A. suum and B. malayi were characterized to provide information on the cause. Maximal response to acetylcholine did not change abruptly, consistent with a discrete event, but rather decreased progressively from A. suum through Dracunculus medinensis, Gonglylonema pulchrum and Thelazia callipaeda. Receptor responses to the characteristic nicotine, and other agonists were generally similar. The decrease in maximal current did correlate with a delayed time to reach larger response. Together, this suggested that the failure to reconstitute the B. malayi N-AChR was one extreme of a progressive decrease and that an issue with synthesis of the receptor in oocytes was responsible. Addition of accessory proteins EMC-6, NRA-2 and NRA-4, in addition to RIC-3, produced a small, but measurable B. malayi N-AChR response. Pharmacological properties of a chimeric B. malayi N-AChR were equivalent to the other species, confirming the receptor response remains unchanged while its production is increasingly dependent on accessory proteins. One possibility is that loss of many subunits for acetylcholine receptors from the filarial nematode genome is linked to new subunit combinations that lead to such a dependence. This novel phylogenetic approach allowed the first characterization of a B. malayi AChR ex vivo and in doing so, provides a framework for the successful characterization of other receptors that have yet to be reconstituted.

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“…ACh leads to a slow increase in baseline movement after prolonged exposure (24 and 48 hours), attributable to inefficient penetration of the lipid cuticle [69] (Figure 1C). Treatment with nicotinic compounds (NIC and LEV) leads to an immediate drop in female and male (10 µM and 100 µM) worm motility followed by quick recovery, mediated by fast responding nAChRs [70]. Treatment with compounds associated with muscarinic activity (ATR, ARE, CAR, OXO) elicit a range of subtle-to-large effects on motility.…”

Section: Resultsmentioning

confidence: 99%

Pharmacological profiling of a Brugia malayi muscarinic acetylcholine receptor as a putative antiparasitic target

Gallo

Wheeler

Elmi

et al. 2022

Preprint

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The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor (Bma-GAR-3) that is highly expressed across intra-mammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Lastly, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

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Anthelmintic resistance and homeostatic plasticity (Brugia malayi)

Cited by 9 publications

References 60 publications

Reconstitution of an N-AChR from Brugia malayi

Reconstitution of an N-AChR from Brugia malayi

Reconstitution of an N-AChR from Brugia malayi, an evolved change in acetylcholine receptor accessory protein requirements in filarial parasites

Pharmacological profiling of a Brugia malayi muscarinic acetylcholine receptor as a putative antiparasitic target

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