State Key Laboratory of Agricultural Microbiology, Key Laboratory for the Development of Veterinary Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
Melbourne Veterinary School, Department of Veterinary Biosciences, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria, 3010, Australia.
Parasit Vectors. 2020 Apr 3;13(1):164. doi: 10.1186/s13071-020-04034-0.
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. 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.
Based 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.
The 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.
These 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.
Smad 蛋白是转化生长因子-β（TGF-β）超家族中细胞内信号转导的重要介质。它们直接将细胞表面受体的传入信号传递到细胞核。尽管 Smad 蛋白具有重要的功能，但几乎没有关于寄生线虫（包括羊捻转胃虫，一种重要的反刍动物吸血线虫）中 Smad 蛋白的信息。
根据羊捻转胃虫的基因组和转录组数据，并使用生物信息学方法，从羊捻转胃虫中推断出 Smad 同源物（称为 Hco-daf-8），并建立了该基因及其编码蛋白 Hco-DAF-8 的结构特征。使用实时 PCR 和免疫荧光检测，研究了 Hco-daf-8 的时空转录和空间表达谱。然后在秀丽隐杆线虫中进行基因拯救，以评估 Hco-daf-8 的功能，并使用人 Smad3 的特异性抑制剂（称为 SIS3）评估 Hco-DAF-8 在羊捻转胃虫发育中的作用。
Hco-DAF-8（502 个氨基酸）的特征，包括保守的 R-Smad 结构域和决定途径特异性的 L3 环残基，与 TGF-β Ⅰ型受体激活的 R-Smad 一致。在所研究的羊捻转胃虫的所有发育阶段均转录 Hco-daf-8 基因，其中第四期幼虫（L4）雌性转录水平较高，成年雄性转录水平最高。Hco-DAF-8 在成虫的盘尾肌细胞、肠道和生殖系统中表达。基因拯救实验表明，Hco-daf-8 能够在秀丽隐杆线虫的 daf-8 缺陷突变株中部分挽救基因功能，导致正常发育的恢复。在羊捻转胃虫中，SIS3 被证明可以影响体外从刚蜕皮的第三期幼虫（L3）到 L4 的发育。
这些发现表明，由基因 Hco-daf-8 编码的 Hco-DAF-8 是 H. contortus 发育的 TGF-β 信号通路中的重要细胞内介质。它们为进一步研究 Hco-DAF-8 及其在 H. contortus 和其他重要寄生线虫中的相关途径提供了基础。
