| Haemonchus contortus is a gastrointestinal parasitic nematode that causes major economic losses resulting from hemorrhagic gastritis,anemia,edema and associated complications in small ruminants.Because of lacking effective vaccine,anthelmintic drugs are unavoidable but good choices to treat the haemonchosis.However,the uncontrolled use of anthelmintics caused serious drug residue and drug resistant problems worldwide.Therefore,there is an urgent need to explore molecular mechanisms controlling the development and reproduction of this parasite in order to develop new drugs and/or vaccines.The dauer hypothesis proposed that iL3 larvae of parasitic nematodes and dauer larvae of free-living nematode Caenorhabditis elegans have many similarities in morphology,growth development and molecular mechanisms in which four signaling pathways are involved,including cyclic guanosine monophosphate?GMP?signaling pathway,dauer transforming growth factor??TGF??pathway,insulin/insulin-like growth factor 1?IGF-1?-like signaling pathway and steroid hormone pathway,which provides a new way to explore the developmental mechanism in parasitic nematodes.It is confirmed that an insulin/IGF-1 like signaling pathway,regulating dauer development in C.elegans,is present in H.contortus,and its components such as the insulin-like receptor Hc-DAF-2,the PI3 kinase Hc-AGE-1 and Hc-AAP-1,3-phosphoinositide dependent protein kinase Hc-PDK-1 and head transcription factor Hc-DAF-16 could regulate the development of H.contorrus,which is supporting evidence for the“dauer hypothesis”.However,nothing is known about the TGF?pathway in H.contortus.In the present study,I isolated three genes?TGF?ligand Hc-tgh-2,TGF?I receptor Hc-tgfbr1 and TGF?II receptor Hc-tgfbr2?of TGF?signaling pathway by molecular methods and bioinformatic technology.Transcriptional levels of the three genes were also assessed by RNAseq.Transgenic technology was used to study the expression patterns in C.elegans N2strain and gene rescue assay was applied to assess the complementation of Hc-tgfbr1 in C.elegans daf-1?DR40?mutant.Recombinant proteins expressed in Escherichia coli were used to immunize rabbits and polyclonal antibodies were produced to detect the localization of these three proteins in H.contortus adults.A TGF?type I receptor inhibitor LY2157299 and RNAi method were used to uncover the importance of the three genes in the developmental processes of H.contortus.?1?The structural and functional characterization of TGF?I receptor?Hc-tgfbr1?The full-length genomic DNA sequence of Hc-tgfbr1 was 4,998 bp in length and had 16exons and 15 introns.The full-length coding sequence of Hc-tgfbr1 was 1,779 bp in length and encoded a protein?Hc-TGFBR1?of 592 amino acids.Phylogenetic analyses showed that Hc-TGFBR1 was a member of TGF?I receptor family and the alignment of homologues showed that Hc-TGFBR1 contains conserved functional domains both in the extracellular region including cysteine residues and intracellular regions including serine/threonine kinase domains and a glycine-serine rich sequence?GS domain?which is characteristic of TGF?type I receptors.Hc-tgfbr1 was transcribed in all developmental stages of H.contortus and with highest levels in parasitic females and iL3s.Treatment of LY2157299 and Hc-tgfbr1 specific dsRNA on exsheathed L3?xL3?retarded the development of xL3 to L4.The immunohistochemical result indicated that Hc-TGFBR1 was expressed in the intestine,exocuticle,and gonad of H.contortus adults.Transgenesis was used in assessing expression patterns and gene rescue assay,finding that Hc-tgfbr1 was expressed in partial intestine?int I and int IX?in C.elegans N2 strain and Hc-tgfbr1 showed no rescue of Ce-daf-1's function in C.elagans daf-1?DR40?mutant.?2?The structural and functional characterization of TGF?II receptor?Hc-tgfbr2?The full-length genomic DNA sequence of Hc-tgfbr2 was 16,150 bp in length and had 16exons and 15 introns.The full-length coding sequence of Hc-tgfbr2 was 1,998 bp in length and encoded a protein?Hc-TGFBR2?of 665 amino acids.Phylogenetic analyses showed that Hc-TGFBR2 was a member of TGF?II receptor family and alignment of homologues showed that Hc-TGFBR2 contains conserved functional domains both in the extracellular region including cysteine residues formed a representative structure?CXCX4C?of TGF?II receptor and intracellular regions including serine/threonine kinase domains.Hc-tgfbr2 was continuously transcribed in all developmental stages of H.contortus and with highest levels in iL3 and male adults.RNAi caused the significant decrease in both Hc-tgfbr2 transcript abundance and the rate of development of xL3 to L4.The immunohistochemical result indicated that Hc-TGFBR2 was expressed in the intestine and gonad of H.contortus adults.Transgenesis was used and found that Hc-tgfbr2 was expressed in intestine and posterior bulb of pharynx in C.elegans N2 strain.?3?The structural and functional characterization of TGF?ligand?Hc-tgh-2?The full-length genomic DNA sequence of Hc-tgh-2 was 14,938 bp in length and had 10exons and 9 introns.The full-length coding sequence of Hc-tgh-2 was 1,050 bp in length and encoded a protein?Hc-TGH-2?of 349 amino acids.Phylogenetic analyses showed that Hc-TGH-2 was a member of TGF?ligand family and alignment of homologues showed Hc-TGH-2 has conserved TGF?2 ligand domain containing conserved cysteine residues.Hc-tgh-2 was continuously transcribed at low level in all developmental stages of H.contortus and with highest levels in iL3.RNAi caused the significant decrease in both Hc-tgh-2 transcript abundance and the rate of development of xL3 to L4.The immunohistochemical result indicated that Hc-TGH-2 was expressed in intestine,exocuticle,and gonad of H.contortus adults.The present study was the first time to explore the structure and function of three important genes from TGF?signaling pathway in H.contortus.This not only helps us to understand the developmental progress of H.contortus,particularly in the transition from free-living stage to parasitic stages,but also provides new information for development of novel drugs and/or vaccines. |
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