Identification and Characterization of Eimeria tenella Microneme Protein (EtMIC8).

Abstract

ABSTRACTMicroneme proteins (MICs) of Eimeria tenella play key roles in motility, migration, attachment, and invasion processes. More than 20 apicomplexan parasite’s MICs have been identified, with nine Eimeria MICs being reported. In this study, a novel E. tenella MIC was identified, and its gene structural features, developmental expression levels, localization, role in adhesion and invasion, and immunogenicity were studied. The results showed that the open reading frame was 1,650 bp, encoding 550 amino acids. It contains a signal sequence, a transmembrane region, four low-complexity boxes, and five epidermal growth factor-like domains (EGF). Subcellular localization revealed its distribution on the membrane surface of the parasite. These characteristics are consistent with the common features of MICs and are named EtMIC8. Anti-EtMIC8 antibodies recognized a specific binding of about 100 kDa in E. tenella, which was twice as large as the prokaryotic expression (about 50 kDa), suggesting that MIC8 may exist naturally as a dimer. EtMIC8 was expressed at higher levels in sporozoites (3.08-fold) and merozoites (2.1-fold) than in sporulated oocysts. The attachment assays using a yeast surface display of MIC8 and its different domains showed that the adherence rates of EtMIC8 to host cells were significantly higher than those of the control (3.17-fold), which was the full contribution of EGF, but neither was alone. Anti-EtMIC8 antibodies significantly reduced the invasion rate of sporozoites into host cells compared to those of the control (P < 0.01). Recombinant EtMIC8-EGF peptides could provide moderate protective efficacy (anticoccidial index [ACI]: 169.7), induce humoral responses, and upregulate CD3+CD8+ lymphocyte cells. IMPORTANCE Microneme is a conserved and specialized apical secretory organelles of apicomplexan parasite. Proteins secreted by microneme (MICs) distribute over the surface of parasites when contacted with the host cells and play important roles in parasites’ gliding motility, migration, adhesion, and host cell invasion. Although numerous MICs of the apicomplexan were reported, only nine E. tenella MICs have been identified, and more work need to be done. The work reported here newly identifies a microneme protein of E. tenella, which plays a role in adhesin and invasion process and has good immunogenicity. These results will provide theoretical support for further understanding of the invasion mechanism of E. tenella and provide data support for the development of subunit vaccines.