Division of Veterinary Infection Biology and Immunology, Research Center Borstel, Borstel, Germany.
Division of Microbial Interface Biology, Research Center Borstel, Borstel, Germany.
Transbound Emerg Dis. 2020 Mar;67 Suppl 1:40-55. doi: 10.1111/tbed.13458.
Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T. annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T. annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.
白细胞增生性泰勒虫寄生虫具有将其牛宿主细胞转化为肿瘤样特征的独特能力，如不受控制的增殖。这种寄生虫依赖性过程的分子机制知之甚少。在目前的研究中，对来自不同泰勒虫分离株的泰勒虫表面蛋白（TaSP）进行生物信息学分析，在 TaSP 的细胞外多态域中鉴定出一个保守的 CDK1 磷酸化基序 TPTK。用放射性标记的 ATP 进行磷酸化测定以及使用磷酸苏氨酸-脯氨酸（pThr-Pro）抗体的 ELISA 实验表明，CDK1-周期蛋白 B 特异性磷酸化 T ，鉴定 TaSP 为体外的底物。共聚焦显微镜和接近连接测定表明 CDK1 和 TaSP 之间在感染了泰勒虫的细胞中相互作用。进一步的研究表明，pThr-Pro 信号和 TaSP 的几乎完全共定位仅发生在有丝分裂期的细胞中，这表明这是一个细胞周期依赖性事件。对分离的、未通透的裂殖子进行免疫染色证实了 pThr-Pro 表位存在于裂殖子的表面。λ磷酸酶处理消除了裂殖子的 pThr-Pro 信号，该信号通过添加 CDK1-周期蛋白 B 得以重建。用 CDK1 抑制剂 purvalanol A 处理感染了泰勒虫的细胞会导致细胞形态发生变化，表现为微管蛋白丰富的细胞突起和裂殖子的延伸，并且 BrdU 掺入和 Ki67 染色的感染了泰勒虫的细胞呈剂量依赖性减少，这表明对宿主细胞 CDK1 的依赖性增殖有明显的影响。我们的数据揭示了寄生虫表面蛋白 TaSP 是宿主细胞激酶 CDK1 的靶标，CDK1 是细胞分裂过程中的主要参与者。针对感染了泰勒虫的细胞的不受控制的增殖是一种限制寄生虫负荷的新的合理方法，以促进针对寄生虫的成功的细胞免疫反应。
