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 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). 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 (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. Results: The 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. 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 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.

Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion

“…At 48 h pi, EteIF-5A was mainly localized in the cytoplasm of immature schizonts, and fluorescence was weaker (Figure 8E and Supplementary Figure 3). At 72 h pi, EteIF-5A was mainly localized in the cytoplasm of mature schizonts (Li C. et al, 2020), and fluorescence was stronger than at 48 h pi (Figure 8F).…”

Eimeria proteins: order amidst disorder

“…By extension, MIC 1–5, 7–9 and apical membrane antigen (AMA) 1–2 have been identified in sporozoites of E. tenella [ 41 , 71 , 85 , 90 , 91 ] with several MIC and AMA orthologues [ 92 ]. Similarly, MIC 1, 2, 4, 5 and 7 and AMA 2 have been identified in E. tenella second-generation merozoites [ 93 ].…”

“…However, ESP is a non-micronemal protein expressed on the surface of permeabilised sporozoites, sporocysts and second-generation merozoites of E. tenella (Table 1 ). Using glutathione S-transferase fusion protein pull-down and bimolecular fluorescence complementation assays, ESP was shown to directly interact with AMA1 of E. tenella to mediate sporozoite invasion [ 92 ] but the regulatory, phenotypic and genetic consequences of AMA1/ESP complex were not completely elucidated as authors only suggested post-translational modification of these proteins. Similarly, Eimeria -conserved protein (ECP) is specific to E. maxima , E. acervulina and E. tenella but its expression is most prominent in sporozoites of E. tenella [ 110 ].…”

“…Protein interactions can affect diverse cellular functions [ 92 ] but protein size is de facto insufficient and limiting (Table 1 ) except if converted to a peptide sequence [ 145 ]. Meanwhile, obvious challenges with mass spectrometry include decoy search strategy [ 146 ], correct peptide identification [ 91 , 112 ] and intractable genome annotations [ 144 ].…”

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

“…The genome of avian species E. tenella comprises approximately 60 Mbp DNA ( Shirley, 2000 ), with approximately 8,618 genes ( Cai et al, 2003 ). Over the past decades, several techniques such as immunofluorescence localization, recombinant proteins, Western blotting, RT-PCR, and protein pull-down assay have been used to delineate the function of genes for cell cycle ( Diallo et al, 2019 ), invasion ( Li et al, 2020a , b ), and drug resistance ( Yu et al, 2021 ). However, due to the lack of genome manipulation tools, the function of only a very few genes has been well deciphered ( Blake, 2015 ).…”