Identification And Functional Characterization Of R-Smads In Haemonchus Contortus

Haemonchus contortus is one of the most important parasitic nematodes in the gastrointestinal tract of ruminants such as cattle and sheep.This nematode feeds on blood,secretes toxins and causes mechanical damage which causes infective animal anemia,emaciation,diarrhea,often leading to the death of severely affected animals.Due to the lack of effective vaccines in China,the poor control effect of existing drugs and the emergence of drug resistance,efficient new vaccines and drugs are urgently needed.With the development of molecular biology,the publication of genome and transcriptome data of H.contortus provides an effective basis for the studies of the growth and development of this nematode and the molecular mechanism of its pathogenesis,however,some scientific problems such as the regulatory mechanism,invasion and immune evasion mechanism of infected larvae of H.contortus have not been solved well which are seriously hampering the development of new anti-parasite drugs or vaccines.Based on similarities in behavior,morphology,and development between the infective third-stage larvae of parasitic nematodes and Caenorhabditis elegans dauer larvae,it is proposed that the regulatory mechanism of dauer development may also regulate the development of the infective third-stage larvae of parasitic nematodes.Studies have shown that there are similar regulatory pathways in H.contortus which phylogenetically belongs to the same branch(V branch)as C.elegans.In H.contortus,the important components of the TGF-? signaling pathway-ligand Hc-DAF-7,type I receptor Hc-TGFBR1,type II receptor HcTGFBR2,Co-Smad Hc-DAF-3 and transcription factor Hc-DAF-5 were identified and were proved to be important for the development from the infective third larval stage to the fourth stage in vitro.R-Smads,as important regulators of the TGF-? signaling pathway,play a crucial role in this pathway of C.elegans,but nothing is known in parasitic nematodes.Based on the protein sequences of R-Smads(Ce-DAF-8,Ce-DAF-14,and Ce-SMA-2)in C.elegans,I searched the genome and transcriptome data of H.contortus and found three corresponding homologous genes and named Hc-daf-8,Hc-daf-14 and Hc-sma-2.Amino acid sequence alignment and phylogenetic tree analysis showed that Hc-DAF-8 and Hc-SMA-2 have conserved functional domains and specific amino acid residues of R-Smad protein and Hc-DAF-8 belongs to the TGF-?-activated R-Smads subfamily,whereas HcSMA-2 belongs to the BMP-activated R-Smads subfamily.Both Hc-DAF-14 and Ce-DAF-14 lack of N-terminal conserved MH1 domain.The two amino acids that determine the specificity of the R-Smad subtype in the L3 loop of Hc-DAF-14 are consistent with TGF-?-activated R-Smads,but phylogenetic tree showed that both of them are not grouped with the two common types of R-Smad.Transcriptional levels of Hc-daf-8,Hc-daf-14 and Hcsma-2 genes in eight developmental stages and sex(egg,L1 larvae,L2 larvae,L3 larvae,L4 male,L4 female,adult female and adult male)were analyzed by fluorescence quantitative PCR.The results showed that these three genes were transcribed at all developmental stages,of which Hc-daf-8 and Hc-sma-2 had the highest transcription levels in the adult male stage,and Hc-daf-14 had the highest transcription levels in the L3 and adult stages.Immunofluorescence assay was carried out on the adults of H.contortus with the prepared effective multi-antibody serum to determine the protein expression.The results showed that Hc-DAF-8 was expressed in the platymyrian muscle cells,intestinal cytoplasm and some eggs of the adult female and the platymyrian muscle cells,intestine and cement glands of the male.Hc-SMA-2 was mainly expressed in the platymyrian muscle cells,eggs of female adults and testis,intestines of the adult male.Functional studies showed that Hc-daf-8 can rescue the mutant strain of Ce-daf-8 and make them develop normally to adults.SIS3,a specific inhibitor of human Smad3,has also been shown to significantly inhibit the development from the infective third larval stage to the fourth stage in vitro.Besides,RNAi assay showed that specific si RNA effectively knocked down the transcription levels of Hc-daf-14 and Hc-sma-2,and significantly reduced the development rate from the infective third larval stage to the fourth stage in vitro by RNAi of Hc-sma-2.In this study,R-Smad encoding genes Hc-daf-8,Hc-daf-14,and Hc-sma-2,as important components of the TGF-? signaling pathway of H.contortus,were identified for the first time,which improved the studies on TGF-? signaling pathway in H.contortus.The functions of Hc-daf-8 and Hc-sma-2 are consistent with other components of the TGF-? signaling pathway,playing a vital role in the development from the infective third larval stage to the fourth stage in vitro in this nematode.Following the systematic studies on the insulin signaling pathway of H.contortus,the systematic studies on the TGF-? signaling pathway provide more theoretical basis for the studies on the regulatory mechanism of H.contortus infectious third larvae concerning invasive host and also provides some theoretical guidance for the development of new and efficient drug targets.