title: Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella.
abstract: 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 (EtCDPK4), 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 EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 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 EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Ziwen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.|Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, PR China.
authors.forename: Bing
authors.initials: B
authors.lastname: Huang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Dong
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Qiping
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Shunhai
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Weili
authors.initials: W
authors.lastname: Xia
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Shuaibin
authors.initials: S
authors.lastname: Xu
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Cui
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Tang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Qifei
authors.initials: Q
authors.lastname: Men
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Zhiyuang
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Cong
authors.initials: C
authors.lastname: Li
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Xuelong
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Han
mesh_terms: D000913:Antibodies, Protozoan / Q000276:immunology; D003001:Cloning, Molecular; D016786:Eimeria tenella / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D000913:Antibodies, Protozoan; D011494:Protein Kinases; D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases