Evidence of a M1-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

Genome-Wide Analysis of the Phosphoinositide Kinome from Two Ciliates Reveals Novel Evolutionary Links for Phosphoinositide Kinases in Eukaryotic Cells

Background: The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs) that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. Methodology/Principal Findings: We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. Conclusions/Significance: Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs) not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory pathways of phosphoinositides may be more widespread than previously thought in unicellular organisms