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Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation.

ABSTRACT: BACKGROUND: Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis. METHODS: Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively. RESULTS: A 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts. CONCLUSIONS: The gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.

SUBMITTER: Liu D

PROVIDER: S-EPMC3896727 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation.

Liu Dandan D Cao Liqin L Zhu Yulan Y Deng Changjing C Su Shijie S Xu Jinjun J Jin Wenjie W Li Jingui J Wu Lili L Tao Jianping J

Parasites & vectors 20140115

<h4>Background</h4>Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis.<h4>Methods</h4>Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The ...[more]

PMID: 24428893

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Project description:BackgroundThe durable oocyst wall formed from the contents of wall-forming bodies (WFBs) protects Eimeria parasites from harsh conditions and enhances parasite transmission. Comprehending the contents of WFBs and proteins involved in oocyst wall formation is pivotal to understanding the mechanism of the oocyst wall formation and the search for novel targets to disrupt parasite transmission.MethodsTotal proteins extracted from WFBs and the oocyst wall of Eimeria necatrix were subjected to comparative proteomic analysis using tandem mass tag in conjunction with liquid chromatography tandem-mass spectrometry techniques. After functional clustering analysis of the identified proteins, three proteins, including E. necatrix disulfide isomerase (EnPDI), thioredoxin (EnTrx) and phosphoglycerate kinase (EnPGK), were selected for further study to confirm their potential roles in oocyst wall formation.ResultsA total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively. Among these proteins, 1102 were identified as differentially expressed proteins, of which 506 were upregulated and 596 downregulated in the oocyst wall compared to the WFBs. A total of 108 proteins, including compositional proteins of the oocyst wall, proteases, oxidoreductases, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport, were proposed to be involved in oocyst wall formation. The approximate molecular sizes of native EnPDI, EnTrx and EnPGK proteins were 55, 50 and 45 kDa, respectively. EnPDI was present in both type 1 and type 2 WFBs, EnTrx was present only in type 2 WFB2 and EnPGK was present only in type 1 WFBs, whereas all of them were localized to the outer layer of the oocyst wall, indicating that all of them participate in the formation of the oocyst wall.ConclusionsTo the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix. The data obtained from this study form a basis for deciphering the molecular mechanisms underlying oocyst wall formation of Eimeria parasites. They also provide valuable resources for future studies on the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission.

Dataset Information

Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation.

Publications

Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation.

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