Abstract
BackgroundAvian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species. These parasites have complex and diverse life-cycles that require 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).MethodsBimolecular 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 (IFA). An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites.ResultsThe interaction between EtAMA1 and EtEsp was confirmed with BiFC and by GST pull-down. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life-cycle. IFA 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.ConclusionIn this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in sporozoite invasion of host cells and is maybe involved in the growth of the parasite. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.
Highlights
Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species
A BLAST search of the E. tenella genome database showed that the open reading frame (ORF) sequence shared 100% sequence identity with ETH_00016590, which encodes an Eimeria-specific protein, on supercontig Eth_scaff124: 9216–10141
The results showed E. tenella Eimeria -specific protein (EtEsp) was mainly located on the surface of sporozoites, Eimeria tenella apical membrane antigen 1 (EtAMA1) was distributed throughout the cytoplasm and the membrane of sporozoites except for refractile bodies (Fig. 8)
Summary
Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species These parasites have complex and diverse life-cycles that require invasion of their host cells. Avian coccidiosis is a widespread, economically significant disease of poultry that results in annual global economic losses of approximately $2.4 billion, including both production losses and disease prevention and treatment costs [1] It is an enteric disease caused by several species of the protozoan genus Eimeria, predominantly E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella [2]. Most apicomplexans are obligate intracellular parasites and are characterized by their apical complexes of specialized secretory organelles (micronemes, rhoptries and dense granules) [3] They use actin-based motility coupled to regulated protein secretion from their apical organelles to actively invade host cells [4]. Despite the diversity of their target host cells, they maintain a highly conserved mechanism for this active invasion process [5]
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