Analysis of rhodopsin G protein-coupled receptor orthologs reveals semiochemical peptides for parasite (Schistosoma mansoni) and host (Biomphalaria glabrata) interplay

Abstract

Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections. A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia. Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.

Figure 1

Workflow for identification of shared GPCRs mined from the B. glabrata genome and transcriptome of S. mansoni miracidia.

Figure 2

Characterization of GPCR orthologs shared between B. glabrata and S. mansoni miracidia. (A) Phylogenetic tree analysis of shared GPCRs, where each B. glabrata GPCR clusters with an ortholog receptor from S. mansoni. Bootstrap values support the confidence levels of clades. (B) Heatmap showing expression of shared GPCR- (in TPM) encoding genes in S. mansoni miracidia at 6 h post-hatch and different B. glabrata tissues. The columns represent S. mansoni miracidia biological replicates (1–3) and their mean, as well as Biomphalaria (glabrata) tissues.

Figure 3

Miracidial behaviour assay using buccalin and FMRFa peptides. (A) Representative trajectories of miracidial movement before and after the addition of the buccalin solution (100 µM) to the center of the recording area. Each colour represents one indistinguishable miracidium individual. See Movie S1 for representative assay video. (B) Representative trajectories of miracidia movement before and after the addition of the FMRFa solution (100 µM) to the center of the area. Each colour represents one indistinguishable miracidium individual. See Movie S2 for representative assay video. (C) Graph of time duration of miracidia remaining in the videoing zone, and (D) mean acceleration values, before and after the addition of buccalin and FMRFa.

Figure 4

Biomphalaria glabrata precursor sequence and dot blot assay for buccalin and FMRFa in B. glabrata-conditioned water. (A) Buccalin neuropeptide precursor sequence (B) Dot blot using anti-buccalin on B. glabrata-conditioned water extracts at 10, 1 and 0.1 mg. (C) Buccalin neuropeptide precursor sequence. (D) Dot blot using anti-FMRFa on B. glabrata-conditioned water extracts at 10, 1 and 0.1 g$\mathrm{\mu }$. -ve Control represents purified water extract only. For precursor sequences, yellow—signal peptide, red—putative cleavage sites, green—amidation, grey—bioactive peptides, pink—MS peptide matches.