Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion
Abstract Background: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several species of the protozoan parasite Eimeria. Among these species, E. tenella causes hemorrhagic pathologies and high mortality. These parasites have complex and diverse lifestyles that require the invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp).Methods: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C.The localization of EtEsp was analyzed with an immunofluorescence assay. An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites.Results: The interaction between EtAMA1 and EtEsp was confirmed with BiFC in vivo and by GST pull-down in vitro. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life cycle. An immunofluorescence analysis showed that the EtEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells in vitro. Conclusion: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in the sporozoite invasion of host cells and maybe involved in the growth of the parasite in the host. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.