Homology modelling, molecular dynamics simulation and docking evaluation of ß-tubulin of Schistosoma mansoni.

Schistosomiasis is one of the neglected diseases causing considerable morbidity and mortality throughout the world. Microtubules with its main component, tubulin play a vital role in helminthes including schistosomes. Benzimidazoles represent potential drug candidates by binding ß-tubulin. The study aimed to generate a homology model for the ß-tubulin of S. mansoni using the crystal structure of O visaries (Sheep) ß-tubulin (PDB ID: 3N2G D) as a template, then different ß-tubulin models were generated and two previously reported benzimidazole derivatives (NBTP-F and NBTP-OH) were docked to the generated models, the binding results indicated that both S. mansoni, S. haematobium were susceptible to the two NBTP derivatives. Additionally, three mutated versions of S. mansoni ß-tubulin wild-type were generated and the mutation (F185Y) seems to slightly enhance the ligand binding. Dynamics simulation experiments showed S. haematobium ß-tubulin is highly susceptible to the tested compounds; similar to S. mansoni, moreover, mutated models of S. mansoni ß-tubulin altered its NBTPs susceptibility. Moreover, additional seven new benzimidazole derivatives were synthesized and tested by molecular docking on the generated model binding site of S. mansoni ß-tubulin and were found to have good interaction inside the pocket.

A novel G protein-coupled receptor of Schistosoma mansoni (SmGPR-3) is activated by dopamine and is widely expressed in the nervous system.

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of "orphan" amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs.

Histamine signalling in Schistosoma mansoni: immunolocalisation and characterisation of a new histamine-responsive receptor (SmGPR-2).

In parasitic platyhelminthes, including Schistosoma mansoni, biogenic amines play several important roles in the control of motility, metabolism and reproduction. A bioinformatics analysis of the S. mansoni genome identified approximately 16 full-length G protein-coupled receptors (GPCRs) that share significant homology with aminergic receptors from other species. Six of these sequences are structurally related to SmGPR-1 (formerly SmGPCR), a previously described histamine receptor of S. mansoni, and constitute a new clade of amine-like GPCRs. Here we report the cloning of a second member of this clade, named SmGPR-2. The full-length receptor cDNA was expressed in Saccharomyces cerevisiae and shown to be activated by histamine and 1-methylhistamine, whereas other common biogenic amines had no significant effect. Antagonist assays showed that SmGPR-2 was inhibited by classical biogenic amine antagonists but the pharmacological profile was unlike those of known mammalian histamine receptors. Confocal immunolocalisation studies revealed that SmGPR-2 was expressed in the nervous system and was particularly enriched in the subtegumental neuronal plexus of adult S. mansoni and larvae. The ligand, histamine, was found to be widely distributed, mainly in the peripheral nervous system including the subtegumental plexus where the receptor is also expressed. Finally, SmGPR-2 was shown to be developmentally regulated at the RNA level. Quantitative PCR studies showed it was up-regulated in the parasitic stages compared with cercaria and expressed at the highest level in young schistosomula. The widespread distribution of histamine and the presence of at least two receptors in S. mansoni suggest that this transmitter is an important neuroactive substance in schistosomes.

Developmental expression analysis and immunolocalization of a biogenic amine receptor in Schistosoma mansoni.

A Schistosoma mansoni G-protein coupled receptor (SmGPCR) was previously cloned and shown to be activated by the biogenic amine, histamine. Here we report a first investigation of the receptor's subunit organization, tissue distribution and expression levels in different stages of the parasite. A polyclonal antibody was produced in rabbits against the recombinant third intracellular loop (il3) of SmGPCR. Western blot studies of the native receptor and recombinant protein expressed in HEK293 cells showed that SmGPCR exists both as a monomer (65 kDa) and an apparent dimer of approximately 130 kDa These species were verified by immunoprecipitation of SmGPCR from S. mansoni extracts, using antibody that was covalently attached to agarose beads. Further investigation determined that the SmGPCR dimer was resistant to treatment with various detergents, 4 M urea and 0.1 M DTT but could be made to dissociate at acidic pH, suggesting the dimer is non-covalent in nature. Confocal immunofluorescence studies revealed significant SmGPCR immunoreactivity in sporocysts, schistosomula and adult worms but not miracidia. SmGPCR was found to be most widely expressed in the schistosomula, particularly the tegument, the subtegumental musculature and the acetabulum. In the adult stage we detected SmGPCR immunofluorescence mainly in the tubercles of male worms and, to a lesser extent, the body wall musculature. Localization in sporocysts was mainly confined to the tegument and cells within parenchymal matrices. A real-time quantitative reverse-transcription PCR analysis revealed that SmGPCR is upregulated at the mRNA level in the parasitic stages compared to the free-living miracidium and cercariae, and it is particularly elevated during early sporocyst and schistosomula development. The results identify SmGPCR as an important parasite receptor with potential functions in muscle and the tegument of S. mansoni.

Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology.

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic.

The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies.

The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. Here, we used a bioinformatics approach to identify the proteasomal constituents of the parasitic trematode Schistosoma mansoni. A detailed search of the S. mansoni genome database identified a total of 31 putative proteasomal subunits, including 17 subunits of the regulatory (19S) complex and 14 predicted catalytic (20S) subunits. A quantitative real-time RT-PCR analysis of subunit expression levels revealed that the S. mansoni proteasome components are differentially expressed among cercaria, schistosomula, and adult worms. In particular, the data suggest that the proteasome may be downregulated during the early stages of schistosomula development and is subsequently upregulated as the parasite matures to the adult stage. To test for biological relevance, we developed a transfection-based RNA interference method to knockdown the expression of the proteasome subunit, SmRPN11/POH1. Transfection of in vitro transformed S. mansoni schistosomula with specific short-interfering RNAs (siRNAs) diminished SmRPN11/POH1 expression nearly 80%, as determined by quantitative RT-PCR analysis, and also decreased parasite viability 78%, whereas no significant effect could be seen after treatment with the same amount of an irrelevant siRNA. These results indicate that the subunit SmRPN11/POH1 is an essential gene in schistosomes and further suggest an important role for the proteasome in parasite development and survival.