Identification and characterization of a cDNA encoding a gametocyte-specific protein of the avian coccidial parasite Eimeria necatrix

Liu, Dandan; Wang, Feiyan; Cao, Liqin; Wang, Lele; Su, Shijie; Hou, Zhaofeng; Xu, Jing; Hu, Junjie; Tao, Jianping

doi:10.1016/j.molbiopara.2020.111318

Cited by 7 publications

(15 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The recombinant proteins rEnGAM22 and rEnGAM59 were prepared using a previously published method [22,24]. WFB protein was prepared using the method described below.…”

Section: Generation Of Polyclonal Antibodiesmentioning

confidence: 99%

“…WFB protein was prepared using the method described below. Mouse anti-rEnGAM59 and WFB pAbs were prepared as described previously [22]. Brie y, the 50 µg proteins, which were resuspended in 50 µL phosphate-buffered saline (PBS) and mixed with 50 µL Quick Antibody-Mouse 3W (Biodragon, Beijing, China), were used to immunize 6-week-old BALB/c mice twice following the manufacturer's recommendations.…”

Section: Generation Of Polyclonal Antibodiesmentioning

confidence: 99%

“…The best characterized of these are two tyrosine-rich gametocyte proteins (GAM56 and GAM82) that were identi ed and localized to WFB2 and the inner oocyst wall of Eimeria maxima [13 − 17], E. tenella [17,18], E. acervuline [17] and E. nieschulzi [19]. In addition, a gam56 tmp2 (or gam59) gene that encodes a second GAM56-like protein (GAM59) exists in the genomes of E. tenella [17,20], E. necatrix [21,22] and E. nieschulzi [23]. In our previous study we demonstrated that a speci c antibody to a recombinant version of EnGAM59 recognized the wall-forming bodies in macrogametes and the walls of oocysts in Eimeria necatrix [22].…”

mentioning

confidence: 99%

“…In addition, a gam56 tmp2 (or gam59) gene that encodes a second GAM56-like protein (GAM59) exists in the genomes of E. tenella [17,20], E. necatrix [21,22] and E. nieschulzi [23]. In our previous study we demonstrated that a speci c antibody to a recombinant version of EnGAM59 recognized the wall-forming bodies in macrogametes and the walls of oocysts in Eimeria necatrix [22]. However, it was not clear whether EnGAM59 localized to WFB1 or WFB2.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy

Wang

Liu

Gao

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Background Eimeria parasite infection occurs via ingestion of oocysts. The robust, bilayered 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. The mechanisms of macrogametogenesis and oocyst wall formation in E. necatrix are also unknown. Methods WFBs of E. necatrix were extracted from purified gametocytes using a cut-off filter and observed by negative stain electron microscopy to assess the intactness of the WFBs. The extracts of purified WFBs and gametocytes were analyzed using sodium dodecyl sulphate-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.

show abstract

“…The recombinant proteins rEnGAM22 and rEnGAM59 were prepared using a previously published method [22,24]. WFB protein was prepared using the method described below.…”

Section: Generation Of Polyclonal Antibodiesmentioning

confidence: 99%

Section: Generation Of Polyclonal Antibodiesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy

Wang

Liu

Gao

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Eimeria necatrix is a highly pathogenic pathogen that mainly colonizes the midsegments of the small intestine, causing weight loss, poor feed conversion and high mortality [ 9 ]. This parasite undergoes a complex life-cycle that includes an exogenous phase (sporogony) and an endogenous phase (schizogony and gametogony) [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

et al. 2021

Self Cite

View full text Add to dashboard Cite

Background Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical Abstract

show abstract

Characterization of the novel glucose-methanol-choline (GMC) oxidoreductase EnOXIO1 in Eimeria necatrix

Wang

Zhuang

Liu

et al. 2023

Veterinary Parasitology

View full text Add to dashboard Cite

Identification and characterization of a cDNA encoding a gametocyte-specific protein of the avian coccidial parasite Eimeria necatrix

Cited by 7 publications

References 30 publications

Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy

Examination of gametocyte protein 22 localization and oocyst wall formation in Eimeria necatrix using laser confocal microscopy and scanning electron microscopy

Full-length transcriptome analysis and identification of transcript structures in Eimeria necatrix from different developmental stages by single-molecule real-time sequencing

Characterization of the novel glucose-methanol-choline (GMC) oxidoreductase EnOXIO1 in Eimeria necatrix

Contact Info

Product

Resources

About