Molecular cloning and characterization of a vesicular acetylcholine transporter from onchocerca volvulus and its biochemical characterization in haemonchus contortus.
Onchocerca volvulus is a human subcutaneous parasitic nematode recognized throughout the world as the single most common cause of irreversible blindness. No drug currently available is completely safe and effective for mass treatment against the adult worms. The identification of suitable drug targets is therefore an essential undertaking for onchocerciasis control. Cholinergic neurotransmission depends on the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Vesicular acetylcholine transport is essential for cholinergic neurotransmission because homozygous VAChT knockout mutants in Caenorrhabditis elegans and Drosophila melanogaster do not live for more than a few days. In this study, the O. volvulus putative vesicular acetylcholine transporter (OvVAChT) gene was cloned and characterized. The predicted OvVAChT partial protein is composed of 404 amino acids and contains 11 conserved putative transmembrane domains. The cloned VAChT cDNA forms approximately 75% portion of the gene in comparison to C. elegans unc 17 gene homologue. It contains the 5’ end of the gene and also has 5’ untranslated region which contains 122bp from the adaptor ligated site. The OvVAChT putative protein shows extensive homology to the vesicular acetylcholine transporter, C. elegans unc17 gene (98%). It is also closely related to other vesicular acetylcholine transmitter transporters from Drosophila melanogaster (56%), Anopheles gambiae (50%), Torpedo californica and other Torpedo species (55%) that have been reported. Relative to the amine transporters, the OvVAChT bears a closer relationship to the rat vesicular monoamine transporters VMAT1 (37%) and VMAT2 (39%). In addition, it also displays a weak similarity to a class of bacterial drug resistance and membrane transporters (22%). Alignment of the amino acids for the VAChTs and MATs from various animal species showed high conservation within the transmembrane regions with charged amino acids indicating functional significance of the gene in substrate transportation. Phylogenetic analysis of VAChTs and MATs clustered the Onchocerca gene in the same clade with C. elegans suggesting the close evolutionary relatedness of the two nematodes. The three remaining clades of the neighour-joining tree contain the other invertebrates’ and vertebrates’ VAChTs in two clades and all the monoamine transporters (MATs) in a single clade indicating greater conservation of MATs compared to VAChTs amongst various animals. The phylogeny tree revealed that the nematodes diverged from the ancenstry much earlier in evolution in relation to other animals. The remaining three clades have close evolutionary relationship and diverged from the ancenstry almost at the same time but much later than the nematodes. The presence of a 70kDa protein band in rat brain and its absence in H. contortus or O. volvulus total protein extracts while blotting using rat anti-VAChT antibodies designed from the C-terminus cloned rat VAChT in Western blotting, conformed with the documented marked variations of the amino acid sequences within this region. Thus, the 3’ end can be used in designing specific anti-VAChT antibodies for different animal species. An additional helminth/nematode species which is parasitic, has now been found to contain a VAChT gene similar to those found in C. elegans (unc17) and other invertebrates and vertebrates which are free living organisms. Since unc17 mutations protect against organophosphorus toxicity and the Torpedo electric lobe provides extremely dense cholinergic innervation to the electric organ, these relationships support a role of VAChT of O. volvulus (OvVAChT), in neurotransmission. This molecular cloning and characterization of (OvVAChT), the C. elegans unc17 gene homologue, will provide unlimited amounts of materials for further studies. To address the potential role in disease and in drug and/or vaccine development, recombinant OvVAChT protein will be prepared so that anti-sera can be raised and used to more precisely define the localization of this protein in the worm. Furthermore, it may be possible that a comparison of the inhibition characteristics of the recombinant O. volvulus and human VAChT proteins may reveal compounds which can specifically inhibit the parasite protein. Alternatively, non-cross-reactive antibodies may be useful in tackling the parasite’s defense systems. In the absence of a suitable animal model for screening compounds with potential macrofilaricidal activity against human onchocerciasis, the availability of viable specimens from nodule digestion for laboratory studies is extremely limited. Vesamicol, 2-(4-Phenyl piperidinol) cyclohexanol (AH5183) is a compound that blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles. It blocks the storage of acetylcholine into synaptic vesicles by binding to the VAChT protein. Consequently, nerve transmissiom across the synapse is inhibited leading to loss of motor activity resulting into paralysis which is characterized by loss of motility of the organisms. This study further characterized the VAChT protein biochemically in the related Haemonchus contortus nematode adult worms that are easier to obtain and maintain in vitro. The effect of vesamicol on motility of H. contortus female adult worms in vitro was assessed, to obtain some insights before going into doing the expensive venture with use of O. volvulus worms. The worms were exposed to various dilutions of the vesamicol inhibitor at concentrations of 0.1mM, 0.3mM, 0.5mM, 0.7mM, 0.9mM and 1mM, in physiological saline at 37oC and worm motility was monitored at 30 minutes intervals for 3 hours. A time-response graph plotted from three representative vesamicol concentrations (0.1mM, 0.5mM and 1mM), revealed a general increase in the mean percent inhibition of worm motility with increasing vesamicol concentration and incubation period. Vesamicol dilutions at 1mM reached an Emax of 100% inhibition of worm motility after 2 hours of incubation, while 0.5mM and 0.1mM vesamicol concentrations never attained 100% inhibition even at the end of the 3-hour incubation period. Analysis of variance between the mean percent inhibitions of worm motility revealed a significant difference in the mean % inhibition caused by the different vesamicol concentrations (p<0.0001) at the different periods of incubation (p= 0.007), suggesting that vesamicol was effective against H. contortus female adult worms in vitro. Both the concentration of vesamicol and incubation period were found responsible for the observed increase in percent inhibitions of worm motility. The results of this study provide the first insights into the effect of VAChT inhibitor in viable organisms.