Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, 200241, PR China; College of Life Sciences, Shanghai Normal University, Shanghai 200234, PR China; Tangshan Center for Quality and Safety Inspection and Research of Agricultural Products, Tangshan 063000, PR China.
Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, 200241, PR China.
Eur J Protistol. 2022 Aug;85:125895. doi: 10.1016/j.ejop.2022.125895. Epub 2022 May 16.
Avian coccidiosis is a common disease caused by Eimeria spp. In the genus Eimeria, the species Eimeria tenella is an obligate intracellular parasite that invades mostly chicken cecal epithelial cells. The 14-3-3 protein is one of the most common adaptor proteins. It is involved in regulating protein phosphorylation and is associated with phosphorylated proteins to regulate signal transduction. Previous reports have shown that 14-3-3 protein has a direct regulatory effect on calcium-dependent protein kinases (CDPKs) activity by interacting with CDPKs. In this study, the characteristics of the E. tenella 14-3-3 protein including transcription and translation analyses, localization in different developmental stages etc were analyzed. The interaction between E. tenella 14-3-3 (Et14-3-3) and E. tenella calcium-dependent protein kinase 4 (EtCDPK4) which is a critical molecule in E. tenella invasion of host cells was verified by Bimolecular Fluorescent Complimentary (BiFC), Co-Immunoprecipitation (co-IP), and Glutathione S-transferase (GST) pull-down. The transcription and translation levels were analyzed using real-time quantitative PCR and western blot. The results showed that the mRNA transcription level of Et14-3-3 was highest in the sporozoite, and the translation level was higher in the unsporulated oocyst than in the other stages. Indirect immunolocalization found that Et14-3-3 was located mainly at the anterior of sporozoites and on the surface of second-generation merozoites. As the sporozoites developed in cells, the fluorescence intensity of Et14-3-3 gradually darkened. BiFC results showed green fluorescence under microscopy in 293T cells co-transfected with pBiFC-VN155-Et14-3-3 and pBiFC-VC155-EtCDPK4. Co-IP and GST pull-down showed that Et14-3-3 interacted with EtCDPK4, which is consistent with the BiFC results. These results indicated that Et14-3-3 had significant interactions with EtCDPK4. Co-localization of Et14-3-3 with EtCDPK4 in sporozoites revealed that they were located in the same position. The secretion assay results indicated that Et14-3-3 was a secreted protein but was not secreted from micronemes. These results lay the foundation for further research on the mechanism of action of EtCDPK4 with Et14-3-3 and the functions of Et14-3-3 in the lifecycle of E. tenella.
鸡球虫病是一种由艾美耳属的艾美耳球虫引起的常见疾病。在艾美耳属中，柔嫩艾美耳球虫是一种专性细胞内寄生虫，主要侵袭鸡盲肠上皮细胞。14-3-3 蛋白是最常见的衔接蛋白之一。它参与调节蛋白磷酸化，并与磷酸化蛋白结合，从而调节信号转导。先前的报告表明，14-3-3 蛋白通过与钙依赖性蛋白激酶（CDPKs）相互作用，对钙依赖性蛋白激酶（CDPKs）的活性具有直接调节作用。在这项研究中，分析了柔嫩艾美耳球虫 14-3-3 蛋白（Et14-3-3）的转录和翻译分析、在不同发育阶段的定位等特征。通过双分子荧光互补（BiFC）、免疫共沉淀（co-IP）和谷胱甘肽 S-转移酶（GST）下拉验证了柔嫩艾美耳球虫 14-3-3（Et14-3-3）与柔嫩艾美耳球虫钙依赖性蛋白激酶 4（EtCDPK4）之间的相互作用，EtCDPK4 是柔嫩艾美耳球虫侵入宿主细胞的关键分子。使用实时定量 PCR 和 Western blot 分析转录和翻译水平。结果表明，Et14-3-3 的 mRNA 转录水平在孢子体中最高，在未孢子化的卵囊中的翻译水平高于其他阶段。间接免疫定位发现 Et14-3-3 主要位于孢子体的前部和第二代裂殖体的表面。随着孢子体在细胞中发育，Et14-3-3 的荧光强度逐渐变暗。BiFC 结果显示，在共转染 pBiFC-VN155-Et14-3-3 和 pBiFC-VC155-EtCDPK4 的 293T 细胞中，在显微镜下观察到绿色荧光。Co-IP 和 GST 下拉表明 Et14-3-3 与 EtCDPK4 相互作用，这与 BiFC 结果一致。这些结果表明 Et14-3-3 与 EtCDPK4 有显著的相互作用。Et14-3-3 与 EtCDPK4 在孢子体中的共定位表明它们位于同一位置。分泌实验结果表明，Et14-3-3 是一种分泌蛋白，但不是从微线体中分泌出来的。这些结果为进一步研究 EtCDPK4 与 Et14-3-3 的作用机制以及 Et14-3-3 在柔嫩艾美耳球虫生命周期中的功能奠定了基础。
