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Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system.

ABSTRACT: Schistosomiasis is an acute and chronic disease caused by parasitic worms of the genus Schistosoma. Treatment is solely dependent on praziquantel. In the face of the worldwide dimension, projects have been initiated to develop new chemotherapies. Due to their proven druggability, G protein-coupled receptors (GPCRs) are promising targets for anthelmintics. However, to identify candidate receptors, a deeper understanding of GPCR signalling in schistosome biology is essential. Comparative transcriptomics of paired and unpaired worms and their gonads revealed 59 differentially regulated GPCR-coding genes putatively involved in neuronal processes. In general, the diversity among GPCRs and their integral membrane topology make it difficult to characterize and deorphanize these receptors. To overcome existing limitations, we performed a pilot approach and utilized the innovative Membrane-Anchored Ligand And Receptor yeast two-hybrid system (MALAR-Y2H) to associate potential neuropeptide ligands with their cognate receptors. Here, we demonstrated the ability to express full-length GPCRs of Schistosoma mansoni in a heterologous yeast-based system. Additionally, we localized GPCRs and chimeras of neuropeptides fused to the WBP1 transmembrane domain of yeast to the plasma membrane of yeast cells. Reporter gene assays indicated ligand-receptor binding, which allowed us to identify certain neuropeptides as potential ligands for two GPCRs, which had been found before to be differentially expressed in schistosomes in a pairing-dependent manner. Thus, the MALAR-Y2H system appears suitable to unravel schistosome GPCR-ligand interactions. Besides its relevance for understanding schistosome biology, identifying and characterizing GPCR-ligand interaction will also contribute to applied research aspects.

SUBMITTER: Weth O

PROVIDER: S-EPMC7284817 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Project description:BackgroundSchistosomiasis is a parasitic disease affecting ~200 million people worldwide. Schistosoma haematobium and S. mansoni are two relatively closely related schistosomes (blood flukes), and the causative agents of urogenital and hepatointestinal schistosomiasis, respectively. The availability of genomic, transcriptomic and proteomic data sets for these two schistosomes now provides unprecedented opportunities to explore their biology, host interactions and schistosomiasis at the molecular level. A particularly important group of molecules involved in a range of biological and developmental processes in schistosomes and other parasites are the G protein-coupled receptors (GPCRs). Although GPCRs have been studied in schistosomes, there has been no detailed comparison of these receptors between closely related species. Here, using a genomic-bioinformatic approach, we identified and characterised key GPCRs in S. haematobium and S. mansoni (two closely related species of schistosome).MethodsUsing a Hidden Markov Model (HMM) and Support Vector Machine (SVM)-based pipeline, we classified and sub-classified GPCRs of S. haematobium and S. mansoni, combined with phylogenetic and transcription analyses.ResultsWe identified and classified classes A, B, C and F as well as an unclassified group of GPCRs encoded in the genomes of S. haematobium and S. mansoni. In addition, we characterised ligand-specific subclasses (i.e. amine, peptide, opsin and orphan) within class A (rhodopsin-like).ConclusionsMost GPCRs shared a high degree of similarity and conservation, except for members of a particular clade (designated SmGPR), which appear to have diverged between S. haematobium and S. mansoni and might explain, to some extent, some of the underlying biological differences between these two schistosomes. The present set of annotated GPCRs provides a basis for future functional genomic studies of cellular GPCR-mediated signal transduction and a resource for future drug discovery efforts in schistosomes.

Project description:The Venus kinase receptor (VKR) is a single transmembrane molecule composed of an intracellular tyrosine kinase domain close to that of insulin receptor and an extracellular Venus Flytrap (VFT) structure similar to the ligand binding domain of many class C G protein coupled receptors. This receptor tyrosine kinase (RTK) was first discovered in the platyhelminth parasite Schistosoma mansoni, then in a large variety of invertebrates. A single vkr gene is found in most genomes, except in S. mansoni in which two genes Smvkr1 and Smvkr2 exist. VKRs form a unique family of RTKs present only in invertebrates and their biological functions are still to be discovered. In this work, we show that SmVKRs are expressed in the reproductive organs of S. mansoni, particularly in the ovaries of female worms. By transcriptional analyses evidence was obtained that both SmVKRs fulfill different roles during oocyte maturation. Suppression of Smvkr expression by RNA interference induced spectacular morphological changes in female worms with a strong disorganization of the ovary, which was dominated by the presence of primary oocytes, and a defect of egg formation. Following expression in Xenopus oocytes, SmVKR1 and SmVKR2 receptors were shown to be activated by distinct ligands which are L-Arginine and calcium ions, respectively. Signalling analysis in Xenopus oocytes revealed the capacity of SmVKRs to activate the PI3K/Akt/p70S6K and Erk MAPK pathways involved in cellular growth and proliferation. Additionally, SmVKR1 induced phosphorylation of JNK (c-Jun N-terminal kinase). Activation of JNK by SmVKR1 was supported by the results of yeast two-hybrid experiments identifying several components of the JNK pathway as specific interacting partners of SmVKR1. In conclusion, these results demonstrate the functions of SmVKR in gametogenesis, and particularly in oogenesis and egg formation. By eliciting signalling pathways potentially involved in oocyte proliferation, growth and migration, these receptors control parasite reproduction and can therefore be considered as potential targets for anti-schistosome therapies.

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Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system.

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