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. 2018:25:60.
doi: 10.1051/parasite/2018061. Epub 2018 Nov 26.

Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella

Ming Yan  1 , Xiaoxia Cui  2 , Qiping Zhao  3 , Shunhai Zhu  3 , Bing Huang  3 , Lu Wang  3 , Huanzhi Zhao  3 , Guiling Liu  3 , Zhihang Li  1 , Hongyu Han  3 , Hui Dong  3
Affiliations

Molecular characterization and protective efficacy of the microneme 2 protein from Eimeria tenella

Ming Yan et al. Parasite. 2018.

Abstract
in English, French

Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.

Les protéines des micronèmes jouent un rôle important dans l’adhésion des parasites Apicomplexa aux cellules hôtes au cours du processus d’invasion. Dans cette étude, la protéine de micronème 2 du protozoaire parasite Eimeria tenella (EtMIC2) a été clonée, caractérisée et son efficacité protectrice en tant que vaccin à ADN a été étudiée. Le gène EtMIC2, qui code pour une protéine de 35.07 kDa dans des oocystes sporulés d’E. tenella, a été cloné et la protéine EtMIC2 recombinante (rEtMIC2) a été produite dans un système d’expression d’Escherichia coli. Une immuno-coloration avec un anticorps anti-rEtMIC2 a montré que la protéine EtMIC2 se localisait principalement dans la région antérieure et la membrane des sporozoïtes, dans le cytoplasme des mérozoïtes de première et de deuxième génération, et était fortement exprimée au cours du premier stade de schizogonie. De plus, une incubation avec des anticorps spécifiques contre EtMIC2 s’est avérée efficace pour réduire la capacité des sporozoïtes d’E. tenella à envahir les cellules hôtes. En outre, des expériences d’infestation ont montré que l’immunisation avec pcDNA3.1(+)-EtMIC2 augmentait de manière significative le gain de poids corporel moyen, tout en diminuant le score de lésion moyen et l’excrétion d’oocystes des poulets. Pris dans leur ensemble, ces résultats suggèrent qu’EtMIC2 joue un rôle important dans l’invasion de cellules parasitaires et pourrait constituer un candidat viable pour la mise au point de nouveaux vaccins contre l’infection à E. tenella chez les poulets.

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Figures

Figure 1
Figure 1
Analysis of EtMIC2 expression in E. coli BL21. (A) Protein expression in bacterial pellets at different times of induction. Lane 1 protein marker; Lane 2 negative control (not induced with IPTG); Lanes 3, 4, 5, and 6 induced with IPTG at 2, 4, 6, and 8 h, respectively. (B) Protein expression in sonicated bacterial cells induced with IPTG at 8 h. S: supernatant, P: precipitate.
Figure 2
Figure 2
Localization of EtMIC2 in sporozoites, second-generation merozoites, and mature first-generation schizonts by indirect immunofluorescence. The parasites were immunostained with anti-rEtMIC2 antibodies, visualized with FITC (green) and counter-stained with DAPI (blue). Abbreviations: pRB, posterior refractile body; N, nucleus; fMz, first-generation merozoites. In vitro sporozoite inhibition assays using recombinant EtMIC2 anti-sera.
Figure 3
Figure 3
In vitro sporozoite inhibition assays using recombinant EtMIC2 anti-sera. Anti-rEtMIC2 stands for IgG purified from recombinant EtMIC2 anti-sera. NA stands for IgG from naive rabbit serum. All assays were performed in triplicate. (*) Indicates that differences between the treatment with anti-rEtMIC2 antibodies and with naive rabbit serum at the same IgG concentration were significant (p < 0.05).
Figure 4
Figure 4
Indirect immunofluorescence assay of EtMIC2 in transfected DF-1 cells. (A, B) pcDNA3.1(+)-EtMIC2-transfected DF-1 cells under fluorescent (FITC) illumination and brightfield, respectively. (C, D) pcDNA3.1(+)-transfected DF-1 cells under fluorescent illumination and brightfield, respectively.
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