DataSheet_1_Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion.xlsx

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

柔嫩艾美耳球虫（Eimeria tenella）是一种顶复门寄生原虫，已知可感染全球范围内的家禽。研究人员已在植物、绿藻、纤毛虫以及顶复门生物（如柔嫩艾美耳球虫）中鉴定出一类重要的钙依赖性蛋白激酶（calcium-dependent protein kinase，CDPK）。CDPK是参与钙信号通路的效应分子，该通路调控滑行运动、繁殖以及宿主细胞入侵等重要生理过程。由于宿主体内不存在CDPK，研究柔嫩艾美耳球虫CDPK的功能可为开发新型治疗药物和疫苗提供理论基础。为探究柔嫩艾美耳球虫CDPK4（EtCDPK4）的功能，研究人员通过免疫共沉淀（co-immunoprecipitation，co-IP）与His标签下拉实验联合质谱分析，筛选出其潜在互作蛋白——翻译起始因子eIF-5A（EteIF-5A）。随后通过双分子荧光互补（bimolecular fluorescence complementation，BiFC）、GST下拉实验以及免疫共沉淀，验证了EteIF-5A与EtCDPK4之间的相互作用，并对EteIF-5A的分子特征进行了分析。本研究采用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction）与蛋白质印迹（western blotting），检测了EteIF-5A在柔嫩艾美耳球虫不同发育阶段的转录与蛋白表达水平。结果显示，EteIF-5A mRNA的转录水平在第二代裂殖子中最高，而其蛋白表达水平在未孢子化卵囊中最高。间接免疫荧光实验结果表明，EteIF-5A蛋白定位于子孢子的整个细胞质中，但不分布于折光体。随着DF-1细胞入侵进程的推进，EteIF-5A的荧光强度在滋养体中升高，在未成熟裂殖体中降低，而在成熟裂殖体中再次升高。经蛋白质印迹分析的分泌实验结果显示，EteIF-5A属于分泌蛋白，但并非由微线体（micronemes）分泌。入侵抑制实验结果表明，兔抗重组EteIF-5A多克隆抗体可有效抑制子孢子的细胞入侵能力，抑制率达48%。