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

Abstract

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.

wrmXpress: A modular package for high-throughput image analysis of parasitic and free-living worms

“…Multivariate phenotyping of Brugia spp. microfilariae and adults (including motility, viability, and fecundity) and Caenorhabditis elegans feeding and development assays are described elsewhere [16,17]. Schistosoma mansoni motility and fecundity assays are described below.…”

“…elegans were maintained using established rearing protocols. Strains N2, unc-122p::GFP, and ZAM11 (gar-3(gk305) V, maz11Ex[myo-2p::Bma-gar-3::unc-54 3'UTR; myo-2p::GFP]) were used for all example images [17]. Transgenic strains were generated using standard microinjection protocols with plasmid DNA [19].…”

“…To our knowledge, a Cellpose model has not been trained for segmenting C. elegans of varying size, shape, and stage. We selected 139 training images from a range of previous experiments [16,17] to include a diverse population of worms, varying in size and shape (straightened or curved with paralytics), and used the Cellpose 2.1.0 GUI to manually segment every worm (>4000 objects). Training images were categorized by the paralytic that was used on the worms (sodium azide or 1P2P) and the sizes of the worms in the image (large, small, or mixed).…”