Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle.
Eimeria tenella protein trafficking: differential regulation of secretion versus surface tethering during the life cycle.
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DOI：
10.1038/s41598-017-04049-1
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发表时间：
2017-07-04
影响因子：
4.6
通讯作者：
Tomley F
中科院分区：
文献类型：
--
作者：
Marugan-Hernandez V;Long E;Blake D;Crouch C;Tomley F
艾美耳球虫属是对家禽有重大影响的细胞内寄生虫。有效的活疫苗是可用的，反向遗传技术的发展提高了利用艾美耳球虫属的前景。作为重组载体来表达另外的免疫保护性抗原。为了研究艾美耳球虫分泌外源抗原或将外源抗原展示在子孢子表面的能力，用瞬时转染法将艾美耳球虫感染子孢子。tenella表达荧光蛋白mCherry，连接到内源性信号肽（SP）和glycophosphatidylinositol锚定（GPI）序列，进行了检查。来自微线蛋白EtMIC2（SP2）的SP允许mCherry有效运输至胞质囊泡，并且在C末端添加GPI锚（来自表面抗原EtSAG1）后，mCherry在子孢子表面上表达。在稳定的转基因群体中，与SP2融合的mCherry被分泌到卵囊的孢子囊腔中，并且在脱囊后，在培养上清液中检测到分泌，但在入侵后没有进入寄生虫空泡。当GPI掺入时，在孢子表面和侵入孢子的上清液中观察到mCherry。经证实的mCherry的分泌和表面暴露表明，与EtSAG1的SP2和GPI的抗原融合物可能是检查针对异源病原体的保护性免疫的诱导的有希望的候选物。
Eimeria spp. are intracellular parasites that have a major impact on poultry. Effective live vaccines are available and the development of reverse genetic technologies has raised the prospect of using Eimeria spp. as recombinant vectors to express additional immunoprotective antigens. To study the ability of Eimeria to secrete foreign antigens or display them on the surface of the sporozoite, transiently transfected populations of E. tenella expressing the fluorescent protein mCherry, linked to endogenous signal peptide (SP) and glycophosphatidylinositol-anchor (GPI) sequences, were examined. The SP from microneme protein EtMIC2 (SP2) allowed efficient trafficking of mCherry to cytoplasmic vesicles and following the C-terminal addition of a GPI-anchor (from surface antigen EtSAG1) mCherry was expressed on the sporozoite surface. In stable transgenic populations, mCherry fused to SP2 was secreted into the sporocyst cavity of the oocysts and after excystation, secretion was detected in culture supernatants but not into the parasitophorous vacuole after invasion. When the GPI was incorporated, mCherry was observed on the sporozites surface and in the supernatant of invading sporozoites. The proven secretion and surface exposure of mCherry suggests that antigen fusions with SP2 and GPI of EtSAG1 may be promising candidates to examine induction of protective immunity against heterologous pathogens.