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, 730046, Gansu, 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. 2020 Feb 27;13(1):105. doi: 10.1186/s13071-020-3975-9.
Anaplasma ovis is a gram-negative, tick-borne obligate intraerythrocytic pathogen, which causes ovine anaplasmosis in small ruminants worldwide. VirB10 of A. ovis is an integral component of the Type IV Secretion System (T4SS). The T4SS is used by bacteria to transfer DNA and/or proteins undeviatingly into the host cell to increase their virulence. To more thoroughly understand the interaction between A. ovis and Dermacentor silvarum, a vector containing the virb10 gene of A. ovis was used as a bait plasmid to screen interacting proteins from the cDNA library of the D. silvarum salivary gland using the yeast two-hybrid system.
The cDNA of the D. silvarum salivary gland was cloned into the pGADT7-SmaI vector (prey plasmid) to construct the yeast two-hybrid cDNA library. The virb10 gene was cloned into the pGBKT7 vector to generate a bait plasmid. Any gene auto-activation or toxicity effects in the yeast strain Y2HGold were excluded. The screening was performed by combining the bait and prey plasmids in yeast strains to identify positive preys. The positive preys were then sequenced, and the obtained sequences were subjected to further analyses using Gene Ontology, UniProt, SMART, and STRING. Additionally, the interaction between the bait and the prey was evaluated using the glutathione S-transferase (GST) pull-down assay.
A total of two clones were obtained from the cDNA library using the yeast two-hybrid system, and the sequence analysis showed that both clones encoded the same large tegument protein, UL36. Furthermore, the proteins GST-UL36 and His-VirB10 were successfully expressed in vitro and the interaction between the two proteins was successfully demonstrated by the GST pull-down assay.
To our knowledge, this study is the first to screen for D. silvarum salivary gland proteins that interact with A. ovis VirB10. The resulting candidate, UL36, is a multi-functional protein. Further investigations into the functionality of UL36 should be carried out, which might help in identifying novel prevention and treatment strategies for A. ovis infection. The present study provides a base for exploring and further understanding the interactions between A. ovis and D. silvarum.
绵羊无形体是一种革兰氏阴性、蜱传的必需红细胞内病原体，它在全球范围内引起绵羊无形体病。绵羊无形体的 VirB10 是 IV 型分泌系统（T4SS）的一个组成部分。细菌利用 T4SS 将 DNA 和/或蛋白质直接转移到宿主细胞中，以增加其毒力。为了更深入地了解绵羊无形体与璃眼蜱之间的相互作用，本研究使用含有绵羊无形体 virb10 基因的载体作为诱饵质粒，通过酵母双杂交系统从璃眼蜱唾液腺 cDNA 文库中筛选互作蛋白。
克隆璃眼蜱唾液腺 cDNA 至 pGADT7-SmaI 载体（诱饵质粒），构建酵母双杂交 cDNA 文库。克隆 virb10 基因至 pGBKT7 载体，构建诱饵质粒。排除酵母菌株 Y2HGold 中任何基因的自动激活或毒性效应。将诱饵和诱饵质粒在酵母菌株中组合进行筛选，以鉴定阳性预诱饵。然后对阳性预诱饵进行测序，并使用 Gene Ontology、UniProt、SMART 和 STRING 对获得的序列进行进一步分析。此外，还通过谷胱甘肽 S-转移酶（GST）下拉实验评估了诱饵和猎物之间的相互作用。
使用酵母双杂交系统从 cDNA 文库中总共获得了两个克隆，序列分析表明这两个克隆均编码相同的大膜蛋白 UL36。此外，体外成功表达了 GST-UL36 和 His-VirB10 两种蛋白，并且通过 GST 下拉实验成功证明了两种蛋白之间的相互作用。
据我们所知，本研究首次筛选到与绵羊无形体 VirB10 相互作用的璃眼蜱唾液腺蛋白。候选蛋白 UL36 是一种多功能蛋白。应进一步研究 UL36 的功能，这可能有助于确定绵羊无形体感染的新的预防和治疗策略。本研究为探索和进一步了解绵羊无形体与璃眼蜱之间的相互作用提供了基础。
