Related Experiment Video
Updated: Aug 3, 2025
Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites
Published on: February 14, 2020
Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal
Lele Wang1,2,3, Dandan Liu4,5,6, Yang Gao1,2,3
1College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, 225009, Jiangsu, People's Republic of China.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
Insights
Eimeria necatrix gametocyte proteins EnGAM22 and EnGAM59 are key to oocyst wall formation. EnGAM22 forms the outer layer via WFB1, while EnGAM59 forms the inner layer via WFB2, clarifying parasite development.
Area of Science:
- Parasitology
- Cell Biology
- Molecular Biology
Background:
- Eimeria parasite infection involves oocyst ingestion.
- Oocyst walls are formed by wall-forming bodies (WFBs) in macrogametocytes.
- Eimeria necatrix gametocyte proteins 22 (EnGAM22) and 59 (EnGAM59) localize to WFBs and the oocyst wall, but their precise locations were unclear.
Purpose of the Study:
- To determine the exact localization of EnGAM22 and EnGAM59 proteins within Eimeria necatrix.
- To elucidate the roles of EnGAM22 and EnGAM59 in the formation of the Eimeria necatrix oocyst wall.
Main Methods:
- Extraction and analysis of WFBs from E. necatrix gametocytes using SDS-PAGE and immunoblotting.
- Indirect immunofluorescence assay to determine protein localization.
- Laser confocal and scanning electron microscopy to analyze macrogametocyte and oocyst wall development.
Main Results:
- Purified WFBs matched in vivo morphology.
- EnGAM22 localized to WFB1, and EnGAM59 localized to WFB2.
- EnGAM22 and EnGAM59 were detected in WFB and gametocyte extracts.
- WFB1 contents formed the outer oocyst wall layer (anti-EnGAM22 positive).
- WFB2 contents formed the inner oocyst wall layer (anti-EnGAM59 positive).
Conclusions:
- EnGAM22 and EnGAM59 are localized to WFB1 and WFB2, respectively.
- These proteins are crucial for forming the outer and inner layers of the E. necatrix oocyst wall.
- Oocyst wall formation in E. necatrix mirrors other Eimeria species; anti-EnGAM22 can track WFB1 protein secretion.
