In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes

Abstract

Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the lead targets. In order to successfully validate NP-GPCRs for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identification of receptor activating ligand(s) via deorphanisation. Such efforts require the identification of all neuropeptide ligands within parasites. Here we mined the genomes of nine therapeutically relevant pathogenic nematodes to characterise the neuropeptide-like protein complements and demonstrate that: (i) parasitic nematodes possess a reduced complement of neuropeptide-like protein-encoding genes relative to Caenorhabditis elegans; (ii) parasite neuropeptide-like protein profiles are broadly conserved between nematode clades; (iii) five Ce-nlps are completely conserved across the nematode species examined; (iv) the extent and position of neuropeptide-like protein-motif conservation is variable; (v) novel RPamide-encoding genes are present in parasitic nematodes; (vi) novel Allatostatin-C-like peptide encoding genes are present in both C. elegans and parasitic nematodes; (vii) novel neuropeptide-like protein families are absent in C. elegans; and (viii) highly conserved nematode neuropeptide-like proteins are bioactive. These data highlight the complexity of nematode neuropeptide-like proteins and reveal the need for nomenclature revision in this diverse neuropeptide family. The identification of neuropeptide-like protein ligands, and characterisation of those with functional relevance, advance our understanding of neuropeptide signalling to support exploitation of the neuropeptidergic system as an anthelmintic target.

Keywords: Ascaris; NLP; Nematode; Neuropeptide; Neuropeptide-like protein; Parasite.

Graphical abstract

Fig. 1

Novel predicted Caenorhabditis elegans neuropeptide-like proteins (Ce-NLPs) are conserved in parasitic nematodes. Multiple sequence alignments were performed using Vector NTI Advance 11.5 AlignX® (Lu and Moriyama, 2004). Text in italics highlights signal peptides as identified using Signal P 4.1 (Nielsen, 2017). Bold text indicates peptides predicted previously (see Van Bael et al., 2018). Bold underlined text indicates novel peptides predicted in this study based on their conservation in additional nematode species. Yellow and blue highlighted regions specify completely and partially conserved amino acid residues, respectively; green specifies similar residues. For all other novel NLP predictions made here (derived from nlp-8, -40, -48, -48, -52, -56, -58, -66, -67, -79 and -81 prepropeptide alignments) see Supplementary Table S5 and Supplementary Fig. S1. Ce, C. elegans; As, Ascaris suum; Na, Necator americanus; Hc, Haemonchus contortus; Bx, Bursaphelenchus xylophilius; Gp, Globodera pallida.

Fig. 2

Clustered Analysis of Sequences (CLANS) clustering supports RPamide and Allatostatin-C like peptide encoding gene nomenclature. (A) Similarity matrix derived from all-against-all BLASTp comparisons between all identified nematode RPamide encoding prepropeptide sequences (E-value limit = 1). (B) Similarity matrix derived from all-against-all BLASTp comparisons between all identified nematode Allatostatin-C like prepropeptide sequences (E-value limit = 1E−5). nlp, neuropeptide-like protein.

Fig. 3

Selected neuropeptide-like protein predicted peptides (NLPs) are bioactive on the Ascaris suum ovijector muscle. (A) Representative muscle tension recordings showing the effect of As-NLP-36, As-NLP-47, As-NLP-49, and As-PDF-1 peptides. Arrows indicate peptide exposure. (B) The effects of NLPs on (i) A. suum ovijector contraction frequency, (ii) contraction amplitude, and (iii) baseline tension. Peptide exposure: 0–10 min. Scale: horizontal bar represents 1 min, vertical bar represents 1 mg.