Evidence of a M-muscarinic GPCR homolog in unicellular eukaryotes: featuring Acanthamoeba spp bioinformatics 3D-modelling and experimentations.

Acetylcholine affects the target cellular function via muscarinic and nicotinic cholinergic receptors that are seen to exist in humans. Both the cholinergic receptors are G-protein coupled receptors (GPCRs) that perform cardinal functions in humans. Anti-muscarinic drugs, particularly the ones that target M1 subtype (mAChR1), have consistently shown to kill unicellular pathogenic eukaryotes like Acanthamoeba spp. As the M1 receptor subtype has not been reported to be expressed in the above protist, the presence of an ancient form of the M1 muscarinic receptor was inferred. Bioinformatic tools and experimental assays were performed to establish the presence of a ligand-binding site. A search for sequence homology of amino acids of human M1 receptor failed to uncover an equivalent ligand-binding site on Acanthamoeba, but structural bioinformatics showed a hypothetical protein L8HIA6 to be a receptor homolog of the human mAChR1. Immunostaining with an anti-mAChR1 antibody showed cellular staining. Growth assays showed proliferation and lethal effects of exposure to mAChR1 agonist and antagonist respectively. With the recent authentication of human mAChR1 structure and its addition to the database, it was possible to discover its structural analog in Acanthamoeba; which could explain the effects of anticholinergics observed in the past on Acanthamoeba spp. The discovery of a receptor homolog of human mAChR1 on Acanthamoeba with future studies planned to show its expression and binding to cholinergic agonist and antagonist would help clarify its role in the biology of this protist pathogen.