The role of Eimeria tenella EtCab protein in the attachment and invasion of host cells
Introduction
Eimeria tenella, a member of the phylum Apicomplexa, is the causative agent of coccidiosis in chicken, which has long been recognized as an economically significant disease in the poultry industry (Yvore et al., 1993; Shirley et al., 2005; Blake et al., 2020). Like other Apicomplexan species, E. tenella is an obligate intracellular parasite. To establish and complete endogenous development of the host cell, attachment and invasion of the host cell are key processes (Sibley, 2010). Many proteins have been reported to be involved in these processes (Augustine, 2001; Lopez-Osorio et al., 2020). The most characterized proteins are apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2), which are released from micronemes and rhoptries into the parasite's membrane and bind directly to the motor complex of the adhesion site (Boucher and Bosch, 2015). Recently, some calcium-binding proteins have also been found to be involved in the invasion process of parasites into host cells. The calmodulin-like calcium binding protein EhCaBP3 of Entamoeba histolytica has been reported to play a role in erythrophagocytosis (Aslam et al., 2012). TgCEN2 (centrin 2, highly conserved calcium-binding protein) and TgCDPK1 (calcium-dependent protein kinase 1) of Toxoplasma gondii have been reported to be essential regulators of calcium-dependent exocytosis, controlling calcium-dependent secretion of micronemes and associated with essential phenotypes, including motility, attachment, invasion and egress of host cells (Lourido et al., 2010; Lentini et al., 2019). The roles of calcium-binding protein of E. tenella in the adhesion and invasion of host cells have not been reported.
In the present study, an E. tenella membrane-associated calcium-binding protein (EtCab) was cloned and analyzed by bioinformatics. Its dynamic expression in different stages of E. tenella, and its role in attachment and invasion of host cells were investigated.
Section snippets
Chickens and parasites
This study was approved by the Animal Care and Use Committee of Shandong Agricultural University (Approval Number: #SDAUA−2019−018). All experiments were performed in accordance with the approved guidelines.
One-day-old Hy-Line Brown cocks were purchased from the hatchery of Hailan (Tai’an, China) and raised under coccidia-free conditions. The E. tenella Shandong strain (SD-01) was isolated, identified and maintained in our laboratory (Liu et al., 2014). Unsporulated oocysts, sporulated oocysts,
Sequence analysis of EtCab
The length of the cDNA sequence was 915 bp, which is 100 % identical to the sequence published in GenBank (XM_013373596). The predicted protein sequence was 304 aa long with a calculated MW of 33,482. Sequence analysis indicated that it has a 24 aa N-terminal signal sequence, followed by 5 complete (I, II, III, IV, V) and one degenerate (VI) EF-hand calcium-binding motifs, as well as the HDEL ER-retention signal at C-terminus (Fig. 1A). Analysis of these indicated that the 5 complete motifs (I,
Discussion
Calcium-binding proteins are responsible for calcium ion transport, which is essential for intracellular signal transduction of the variant physiological activities of eukaryotes (Berridge et al., 2000; Lourido and Moreno, 2015). A major class of calcium-binding proteins is characterized by the presence of a highly conserved helix-loop-helix structural unit, which is known as an EF-hand (Moreno et al., 2011). Calcium-binding proteins containing structural EF-hand motifs include calmodulin
Conclusion
In the present study, an E. tenella EtCab has been cloned and identified. EtCab belongs to the CREC protein family. We have experimentally demonstrated that EtCab localizes on the surface of E. tenella sporozoites and plays a role in the adhesion and the invasion of host cells in vitro.
CRediT authorship contribution statement
Yakun Wang: Investigation, Writing - original draft. Xue Zhou: Investigation, Writing - review & editing. Hanzhu Wang: Investigation. Lingyu Sun: Investigation. Bingxiang Wang: Investigation. Yingying Jiang: Investigation. Huihui Li: Investigation. Xiao Zhang: Methodology. Hongmei Li: Supervision. Xiaomin Zhao: Writing - original draft, Writing - review & editing, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by grants from the National Key Research and Development Program of China (2017YFD0500400), National Natural Science Foundation of China (No. 31572514) and Funds of Shandong Higher Education Youth Science and Technology Support Program (2019KJF022).
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