Open AccessMolecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella
Author(s) -
Ziwen Wang,
Bing Huang,
Hui Dong,
Qiping Zhao,
Shanfeng Zhu,
Weili Xia,
ShangZhong Xu,
Yun Xie,
Xiaoxia Cui,
Min Tang,
Qiu-Lei Men,
Zhiyuang Yang,
Cong Li,
Xinna Zhu,
Hongyu Han
Publication year - 2016
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0168132
Subject(s) - biology , eimeria , in vitro , kinase , complementary dna , polyclonal antibodies , microbiology and biotechnology , protein kinase a , biochemistry , antibody , gene , genetics
Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 ( Et CDPK4), a critical molecule in E . tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E . tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E . tenella . EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether Et CDPK4 was involved in invasion of DF-1 cells by E . tenella sporozoites. Polyclonal antibodies against recombinant Et CDPK4 (r Et CDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored Et CDPK4 distribution during parasite development after E . tenella sporozoite invasion of DF-1 cells in vitro . The results showed that Et CDPK4 might be important in sporozoite invasion and development. To analyze Et CDPK4 functional domains according to the structural characteristics of Et CDPK4 and study the kinase activity of r Et CDPK4, an in vitro phosphorylation system was established. We verified that r Et CDPK4 was a protein kinase that was completely dependent on Ca 2+ for enzyme activity. Specific inhibitors of r Et CDPK4 activity were screened by kinase activity in vitro . Some specific inhibitors were applied to assays of DF-1 cell invasion by E . tenella sporozoites to confirm that the inhibitors functioned in vitro . W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E . tenella sporozoites. The experimental results showed that Et CDPK4 may be involved in E . tenella invasion of chicken cecal epithelial cells.