Dauer signalling pathway model for Haemonchus contortus

Abstract

Background: Signalling pathways have been extensively investigated in the free-living nematode Caenorhabditis elegans, but very little is known about these pathways in parasitic nematodes. Here, we constructed a model for the dauer-associated signalling pathways in an economically highly significant parasitic worm, Haemonchus contortus.

Methods: Guided by data and information available for C. elegans, we used extensive genomic and transcriptomic datasets to infer gene homologues in the dauer-associated pathways, explore developmental transcriptomic, proteomic and phosphoproteomic profiles in H. contortus and study selected molecular structures.

Results: The canonical cyclic guanosine monophosphate (cGMP), transforming growth factor-β (TGF-β), insulin-like growth factor 1 (IGF-1) and steroid hormone signalling pathways of H. contortus were inferred to represent a total of 61 gene homologues. Compared with C. elegans, H. contortus has a reduced set of genes encoding insulin-like peptides, implying evolutionary and biological divergences between the parasitic and free-living nematodes. Similar transcription profiles were found for all gene homologues between the infective stage of H. contortus and dauer stage of C. elegans. High transcriptional levels for genes encoding G protein-coupled receptors (GPCRs), TGF-β, insulin-like ligands (e.g. ins-1, ins-17 and ins-18) and transcriptional factors (e.g. daf-16) in the infective L3 stage of H. contortus were suggestive of critical functional roles in this stage. Conspicuous protein expression patterns and extensive phosphorylation of some components of these pathways suggested marked post-translational modifications also in the L3 stage. The high structural similarity in the DAF-12 ligand binding domain among nematodes indicated functional conservation in steroid (i.e. dafachronic acid) signalling linked to worm development.

Conclusions: Taken together, this pathway model provides a basis to explore hypotheses regarding biological processes and regulatory mechanisms (via particular microRNAs, phosphorylation events and/or lipids) associated with the development of H. contortus and related nematodes as well as parasite-host cross talk, which could aid the discovery of new therapeutic targets.

Keywords: Dauer signalling pathway; Haemonchus contortus; Phospho-proteomic analysis; Proteomic analysis; Transcriptomic analysis.

Fig. 1

Dauer signalling pathway model proposed for Haemonchus contortus. The cGMP (red), TGF-β (orange), insulin-like growth factor 1 (IGF-1) (green) and steroid hormone (light blue) signalling pathways are constructed based on information and data available for C. elegans. Pathway components not identified in H. contortus are indicated in grey. It is hypothesized that environmental signals are received by G-protein-coupled receptors (GPCRs) through cGMP signalling in chemosensory neurons, transduced via TGF-β and insulin-like peptides in parallel TGF-β signalling and IGF-1 signalling, which then converge into the steroid hormone signalling pathway via the biosynthesis of dafachronic acids (DAs; ligands of nuclear hormone receptor DAF-12) in neuroendocrine tissues, determining the activation of DAF-12 in target (hypodermal) cells. The activation of DAF-12 initiates or regulates numerous molecular events such as transcription. Signalling or regulation is indicated by an arrow or cross-line. Phosphorylation events detected in the third larval stage (L3) of H. contortus are indicated by a phosphate group (yellow). Dashed arrows indicate pathways which need to be further elucidated. Drawing is adapted or modified from previous publications [24, 39, 45, 62]

Fig. 2

Transcription of dauer signalling genes in Caenorhabditis elegans and in Haemonchus contortus. Messenger RNA transcriptional levels (fragments per kilobase per million mapped reads, FPKM) of a 60 dauer signalling genes in key developmental stages of C. elegans [egg, the first- (L1), second- (L2), dauer-, fourth- (L4) stage larvae and adults (Ad)], and b all 61 homologous genes in H. contortus stages (egg, L1, L2, L3, L4 (female and male L4s—L4f and L4m), and female and male adults—Adf and Adm) are indicated in the heat maps. Colour scales and Z-scores indicate scaled FPKMs in the rows. Genes involved in the cGMP, TGF-β, IGF-1 and steroid hormone signalling pathways are listed

Fig. 3

DAF-12 sequence and structural similarities among nematodes. a Alignment of the inferred protein sequences of DAF-12 of Haemonchus contortus (Hc-DAF-12), Ancylostoma ceylanicum (Ac-DAF-12; GenBank: EPB79655), Necator americanus (Na-DAF-12; GenBank: XP_013307217), Caenorhabditis elegans (Ce-DAF-12; WormBase ID: CE27585) and Strongyloides stercoralis (Ss-DAF-12; GenBank: AAD37372) is shown, with sequence similarities indicated. Zinc finger domain and ligand binding domain (LBD) are framed in black and red, respectively. b Structure models of LBDs of H. contortus and C. elegans are matched to the published crystal structure (PDB accession no. 3GYU_ChainA; S. stercoralis); TM-scores (0.98 and 0.97) and an overall root-mean-square deviation (RMSD) of 0.83 indicate high pairwise structural similarities