Characterization of the Eimeria tenella rhoptry protein with a nuclear localization sequence (EtROP30)

Abstract

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.

Keywords: Eimeria tenella; EtROP30; Host nucleus; Rhoptry proteins; Vaccine candidate.

Fig. 1

Molecular characterization of EtROP30. a Primary structural analysis of EtROP30; (b) Eight conserved motifs of the E. tenella-specific rhoptry kinase 1 subfamily; (c) Alignment analysis of the amino acid sequences of EtROP30 homologous protein in Eimeria species. Kinase conserved motifs (I-VIII) were framed out with dotted lines. The active sites of protein kinases were marked with asterisks (*); (d) Evolutionary phylogenetic relationship analysis of EtROP30 by MegAlign

Fig. 2

The localization and secretion of EtROP30. a SDS-PAGE analysis of the purified recombinant EtROP30; (b) Western blot analysis of the recombinant EtROP30 recognized by the anti-EtROP30 polyclonal antibody (1:1000); (c) Western blot analysis of the whole sporozoite protein by the anti-EtROP30 polyclonal antibody (1:1000); (d) EtROP30 localization visualized by IFA. Sporozoites and merozoites were labeled with the anti-EtROP30 primary antibody (1:100). Scale bar, 20 μm; (e) Western blot analysis of the whole sporozoite protein and sporozoite secretory protein by the anti-EtROP30 polyclonal antibody (1:1000) and the anti-GAPDH mouse monoclonal antibody (1:6000). GAPDH was used as a reference

Fig. 3

The localizations of EtROP30 and ΔNLS-EtROP30 in DF-1 cells. a Schemes of PEGFP-EtROP30 and PEGFP-ΔNLS-EtROP30; (b) Intracellular localization of EtROP30 and ΔNLS-EtROP30 analyzed by confocal microscopy in DF-1 cells. EGFP fusion proteins were expressed in DF-1 cells and nuclei were labeled by DAPI; (c) Western blot analysis of the cytoplasm and fractions. GAPDH was used as a cytoplasmic reference and Histone H3 as a nuclear reference

Fig. 4

Dynamic expression levels of EtROP30 at different developmental stages of E. tenella. Unsporulated oocysts (UO); sporulated oocysts (SO); sporozoites (SZ); merozoites (MZ) (a) Analysis of mRNA transcription levels of EtROP30 at different developmental stages by qRT-PCR; (b) Western blot analysis of EtROP30 with GAPDH being used as a reference; (c) Quantification of Western blot bands using Image J software. Data represent the mean of triplicate determinations. No significant difference (ns) p > 0.05, significant difference * p < 0.05, ** p < 0.01

Fig. 5

Effective immune protection induced by recombinant EtROP30 against E. tenella. a Western blot analysis of the recombinant EtROP30 recognized by the serum of the challenged control group and the unchallenged control group; (b) Effective immune protection on body weight gain; (c) Relative body weight gain; (d) Cecum lesion score; (e) Number of oocyst excretion; (f) Serum IgY levels. Data are expressed as mean ± SD (error bars) of three independent experiments. All data were analyzed using t-tests. p-values are represented by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001