Developmental expression analysis and RNA interference (RNAi) screen of putative neuromuscular receptors of «Schistosoma mansoni»

In parasitic platyhelminths, including Schistosoma mansoni the coordination of neuromuscular system is critical for continued propagation, development and successful completion of the lifecycle.Neuromuscular signaling in these parasites is mediated by a variety of neurotransmitters, both small molecules ("classical") transmitters and neuropeptides.Biogenic amines (BA) constitute the largest subset of classical neurotransmitters and play several key roles in the control of schistosome muscle function and movement.There are several putative BA receptors identified in the S. mansoni genome, the majority of which are Class A G protein coupled receptors (GPCRs).Here we report the functional role of these putative BA receptors in parasite development and motility by developmental expression analysis and RNAi screening.We performed an expression analysis of several putative BA receptors at the RNA level in different developmental stages of the parasite, using reverse-transcription coupled to quantitative PCR (RT-qPCR).One of these proteins is a previously described serotonin receptor of S. mansoni (named Sm5HTR) and the others are novel "orphan" BA-like receptors.The analysis showed that the BA receptors tested are expressed in all developmental stages, however the majority are preferentially expressed in cercaria and schistosomula, suggesting these receptors play particularly important roles in parasite larvae.Next we performed RNA interference (RNAi) targeting the same BA receptors by transfecting S.mansoni larvae with small interfering RNA (siRNA) and analyzed for effects on motor activity by comparing with the control groups.Given that BAs are known modulators of schistosome movement, we hypothesized that the RNAi would produce a motor phenotype in the larvae and this was confirmed by the data.The results identified strongly hypoactive phenotypes for three out of four receptors tested, including Sm5HTR (Smp_126730), Smp_150180 and Smp_120620), all showing significant reduction in movement compared to control larvae transfected with irrelevant (scrambled) siRNAs.The RNAi phenotype correlated with a significant and specific knockdown in transcript levels as determined by RT-qPCR.To elucidate the mode of action of Sm5HTR we also performed confocal immunolocalization analysis using a specific peptide iii antibody.The expression pattern suggests Sm5HTR is highly abundant in the central and peripheral nervous system of the parasite, including the peripheral innervation of the body wall muscles responsible for movement.Together the results suggest that Sm5HTR and other BA receptors play an important role in the control of schistosome motility, particularly the larvae, and could be potential targets for new drug discovery.

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