Wang Bingxiang,Zhao Ningning,Sun Lingyu,Tan Qianqian,Xiao Qianqian,Chen Junpeng,Li Jinxuan,Zhang Xiao,Zhao Xiaomin
Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella.
Rhoptry proteins (ROPs), secreted by specific rhoptry organelles of apicomplexan parasites, are determinants of parasite pathogenesis and sources of vaccine candidates. Twenty-eight ROPs of Eimeria tenella have been predicted by genomic approaches, and in the present study, E. tenella rhoptry protein 30 (EtROP30) was characterized. Subcellular localizations of EtROP30 in sporozoites and merozoites were in the apical complex and rhoptry-like bulb, suggesting that EtROP30 is a member of ROPs in E. tenella . Sequence analysis showed that EtROP30 contained an N-terminal secretory signal, a protein kinase domain with eight E. tenella -specific rhoptry kinase 1 subfamily (ROPK-Eten1) motifs, and a C-terminal nuclear localization sequence (NLS), making EtROP30 the only ROP that contains both a secretory signal and an NLS in E. tenella . Subsequent experiments showed that EtROP30 was a secreted protein in the sporozoite stage, relying on NLS for migration to the host nucleus. In addition, EtROP30 showed significantly higher expression levels in the parasite merozoite stage, indicating that EtROP30 plays a critical role during parasite reinvasion and development and may be a viable option as a vaccine candidate for anti-parasitic infection. The immunization protection efficacies of EtROP30 were evaluated. Significant improvements in mean body weight gain, reduction of cecum lesion score, and number of oocysts excreted were observed, indicating that EtROP30 has good immunogenicity against E. tenella . In the present study, a ROP of E. tenella with secretory and nuclear localization characteristics has been identified, and proved to be an effective vaccine candidate against this parasite.
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