State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu, 730046, People's Republic of China.
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, People's Republic of China.
Parasit Vectors. 2017 Oct 30;10(1):536. doi: 10.1186/s13071-017-2421-0.
Theileria annulata can infect monocytes/macrophages and B lymphocytes and causes severe lymphoproliferative disease in ruminants. Meanwhile, infection by T. annulata leads to the permanent proliferation of cell population through regulating signaling pathways of host cells. Cysteine proteinases (CPs) are one kind of protein hydrolase and usually play critical roles in parasite virulence, host invasion, nutrition and host immune response. However, the biological function of T. annulata CP (TaCP) is still unclear. In this study, a yeast-two-hybrid assay was performed to screen host proteins interacting with TaCP, to provide information to help our understanding of the molecular mechanisms between T. annulata and host cells.
The cDNA from purified bovine B cells was inserted into pGADT7-SfiI vector (pGADT7-SfiI-BcDNA, Prey plasmid) for constructing the yeast two-hybrid cDNA library. TaCP was cloned into the pGBKT7 vector (pGBKT7-TaCP) and was considered as bait plasmid after evaluating the expression, auto-activation and toxicity tests in the yeast strain Y2HGold. The yeast two-hybrid screening was carried out via co-transforming bait and prey plasmids into yeast strain Y2HGold. Sequences of positive preys were analyzed using BLAST, Gene Ontology, UniProt and STRING.
Two host proteins, CRBN (Bos taurus cereblon transcript variant X2) and Ppp4C (Bos indicus protein phosphatase 4 catalytic subunit) were identified to interact with TaCP. The results of functional analysis showed that the two proteins were involved in many cellular processes, such as ubiquitylation regulation, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs.
This study is the first to screen the host proteins of bovine B cells interacting with TaCP, and 2 proteins, CRBN and Ppp4C, were identified using yeast two-hybrid technique. The results of functional analysis suggest that the two proteins are involved in many cellular processes, such as ubiquitylation regulating, microtubule organization, DNA repair, cell apoptosis and maturation of spliceosomal snRNPs. The interaction with CRBN and Ppp4C indicate that TaCP possibly is involved in regulating signaling pathways and cell proliferation, which is helpful for understanding the interaction between T. annulata and host cells.
环形泰勒虫可感染单核细胞/巨噬细胞和 B 淋巴细胞，并导致反刍动物严重的淋巴增生性疾病。同时，环形泰勒虫感染通过调节宿主细胞的信号通路导致细胞群体的永久性增殖。半胱氨酸蛋白酶（CPs）是一种蛋白水解酶，通常在寄生虫毒力、宿主入侵、营养和宿主免疫反应中发挥关键作用。然而，环形泰勒虫 CP（TaCP）的生物学功能尚不清楚。在本研究中，我们进行了酵母双杂交实验，以筛选与 TaCP 相互作用的宿主蛋白，为我们了解环形泰勒虫与宿主细胞之间的分子机制提供信息。
从纯化的牛 B 细胞中提取的 cDNA 插入 pGADT7-SfiI 载体（pGADT7-SfiI-BcDNA，诱饵质粒）中，构建酵母双杂交 cDNA 文库。将 TaCP 克隆到 pGBKT7 载体（pGBKT7-TaCP）中，经过酵母菌株 Y2HGold 的表达、自激活和毒性测试评估后，将其作为诱饵质粒。通过将诱饵和猎物质粒共转化到酵母菌株 Y2HGold 中进行酵母双杂交筛选。使用 BLAST、GO、UniProt 和 STRING 分析阳性猎物的序列。
鉴定出两种与 TaCP 相互作用的宿主蛋白，CRBN（Bos taurus cereblon 转录变体 X2）和 Ppp4C（Bos indicus protein phosphatase 4 catalytic subunit）。功能分析结果表明，这两种蛋白参与许多细胞过程，如泛素化调节、微管组织、DNA 修复、细胞凋亡和剪接体 snRNPs 的成熟。
本研究首次筛选出与牛 B 细胞中 TaCP 相互作用的宿主蛋白，并用酵母双杂交技术鉴定出 2 种蛋白，CRBN 和 Ppp4C。功能分析结果表明，这两种蛋白参与许多细胞过程，如泛素化调节、微管组织、DNA 修复、细胞凋亡和剪接体 snRNPs 的成熟。与 CRBN 和 Ppp4C 的相互作用表明 TaCP 可能参与调节信号通路和细胞增殖，这有助于理解环形泰勒虫与宿主细胞的相互作用。
