College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China.
Parasit Vectors. 2023 Apr 12;16(1):124. doi: 10.1186/s13071-023-05742-z.
Eimeria parasite infection occurs via ingestion of oocysts. The robust, bilayer oocyst wall is formed from the contents of wall-forming bodies (WFBs), WFB1 and WFB2, located exclusively in macrogametocytes. Eimeria necatrix gametocyte proteins 22 and 59 (EnGAM22 and EnGAM59) have been found to localize to WFBs and the oocyst wall. However, the exact localization of these two proteins is not clear.
WFBs of E. necatrix were extracted from purified gametocytes using a cutoff filter and the extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Then, the localization of EnGAM22 and EnGAM59 proteins was determined using an indirect immunofluorescence assay. Finally, the development of macrogametocytes and the oocyst wall of E. necatrix was analyzed using laser confocal microscopy and scanning electron microscopy.
Purified WFBs had the same shape and size as those observed in macrogametocytes. EnGAM22 protein localized to WFB1, whereas EnGAM59 protein localized to WFB2. Both EnGAM22 and EnGAM59 native proteins were detected in the extracts of WFBs and gametocytes. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface of the macrogametocyte to form an anti-EnGAM22 positive layer. WFB2 then appeared to give rise to the inner layer, which was anti-EnGAM59 positive.
EnGAM22 and EnGAM59 proteins localized to WFB1 and WFB2 and were involved in the formation of the outer and inner layers of the oocyst wall of E. necatrix, respectively. The processes of macrogametogenesis and oocyst wall formation of E. necatrix are similar to other Eimeria parasites. The anti-EnGAM22 antibody could be used as a tool to track the transport and secretion of proteins in WFB1 during oocyst development.
艾美耳球虫寄生虫感染是通过摄入卵囊而发生的。坚固的双层卵囊壁是由仅存在于大配子体中的壁形成体（WFB）WFB1 和 WFB2 的内容物形成的。已经发现艾美耳属球虫配子体蛋白 22 和 59（EnGAM22 和 EnGAM59）定位于 WFB 和卵囊壁。然而，这两种蛋白质的确切定位尚不清楚。
使用截止过滤器从纯化的配子体中提取艾美耳球虫的 WFB，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和免疫印迹分析纯化的 WFB 和配子体提取物。然后，使用间接免疫荧光测定法确定 EnGAM22 和 EnGAM59 蛋白的定位。最后，使用激光共聚焦显微镜和扫描电子显微镜分析艾美耳球虫大配子体和卵囊壁的发育情况。
纯化的 WFB 具有与在大配子体中观察到的相同的形状和大小。EnGAM22 蛋白定位于 WFB1，而 EnGAM59 蛋白定位于 WFB2。EnGAM22 和 EnGAM59 天然蛋白均在 WFB 和配子体提取物中检测到。卵囊壁的外层是通过大配子体表面 WFB1 内容物的释放形成抗 EnGAM22 阳性层而形成的。然后，WFB2 似乎形成了内层，该层为抗 EnGAM59 阳性。
EnGAM22 和 EnGAM59 蛋白定位于 WFB1 和 WFB2，分别参与了艾美耳球虫卵囊壁的外层和内层的形成。艾美耳球虫的大配子体发生和卵囊壁形成过程与其他艾美耳球虫寄生虫相似。抗 EnGAM22 抗体可用作工具来跟踪卵囊发育过程中 WFB1 中蛋白质的运输和分泌。
