Abstract
BackgroundSmad proteins are essential cellular mediators within the transforming growth factor-β (TGF-β) superfamily. They directly transmit incoming signals from the cell surface receptors to the nucleus. In spite of their functional importance, almost nothing is known about Smad proteins in parasitic nematodes including Haemonchus contortus, an important blood-sucking nematode of small ruminants.MethodsBased on genomic and transcriptome data for H. contortus and using bioinformatics methods, a Smad homologue (called Hco-daf-8) was inferred from H. contortus and the structural characteristics of this gene and its encoded protein Hco-DAF-8 established. Using real-time PCR and immunofluorescence assays, temporal transcriptional and spatial expression profiles of Hco-daf-8 were studied. Gene rescue in Caenorhabditis elegans was then applied to assess the function of Hco-daf-8 and a specific inhibitor of human Smad3 (called SIS3) was employed to evaluate the roles of Hco-DAF-8 in H. contortus development.ResultsThe features of Hco-DAF-8 (502 amino acids), including conserved R-Smad domains and residues of the L3-loop that determine pathway specificity, are consistent with a TGF-β type I receptor-activated R-Smad. The Hco-daf-8 gene was transcribed in all developmental stages of H. contortus studied, with a higher level of transcription in the fourth-stage larval (L4) females and the highest level in adult males. Hco-DAF-8 was expressed in the platymyarian muscular cells, intestine and reproductive system of adult stages. Gene rescue experiments showed that Hco-daf-8 was able to partially rescue gene function in a daf-8 deficient mutant strain of C. elegans, leading to a resumption of normal development. In H. contortus, SIS3 was shown to affect H. contortus development from the exsheathed third-stage larvae (L3s) to L4s in vitro.ConclusionsThese findings suggest that Hco-DAF-8, encoded by the gene Hco-daf-8, is an important cellular mediator of H. contortus development via the TGF-β signalling pathway. They provide a basis for future explorations of Hco-DAF-8 and associated pathways in H. contortus and other important parasitic nematodes.
Highlights
Smad proteins are essential cellular mediators within the transforming growth factor-β (TGF-β) superfamily
The alignment of amino acid sequences indicated that all selected receptor-regulated Smad (R-Smad) have a Mad homology 1 (MH1) domain at the N-terminus, which binds DNA and a Mad homology 2 domain (MH2, blue) at the C-terminus, which participates in inter-molecular interactions and regulates transcription [36]
The results showed that Cel-daf8p::Hco-daf-8::gfp was expressed in all life stages, and green fluorescent protein (GFP) expression was mainly detected in the intestine and nervous system (Fig. 5g–l), consistent with the expression patterns in worms transformed with Cel-daf-8p::Celdaf-8::gfp (Fig. 5a–f )
Summary
Smad proteins are essential cellular mediators within the transforming growth factor-β (TGF-β) superfamily They directly transmit incoming signals from the cell surface receptors to the nucleus. The transforming growth factor-β (TGF-β) signalling pathway regulates the growth, development and differentiation of cells in diverse organisms including humans, mice, flies and worms [1, 2] In this pathway, signalling is initiated when the ligand induces the assembly of a heteromeric complex of type II and type I receptors. Following phosphorylation by a type I receptor, R-Smads interact with Co-Smads to form a complex, which is translocated into the nucleus to regulate the transcription of target genes In these processes, R-Smads usually play a central role in maintaining specificity in the TGF-β signalling pathway by functioning both as a substrate of the type I receptor and as an usher for Co-Smad [5]. R-Smads are regulated by inhibitory Smads, which prevent the phosphorylation and/or nuclear translocation of R-Smads [6]
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