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Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella
Ziwen Wang1, Bing Huang1,2, Hui Dong1
1Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai, PR China.
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.
Insights
Calcium-dependent protein kinase 4 (EtCDPK4) is crucial for Eimeria tenella invasion of chicken cells. Inhibiting EtCDPK4 significantly reduced parasite invasion, highlighting its role in host cell entry.
Area of Science:
- Parasitology
- Molecular Biology
- Cell Biology
Background:
- Eimeria tenella is an obligate intracellular parasite causing cecal disease in chickens.
- The molecular mechanisms of E. tenella host cell invasion are not fully understood.
- Calcium-dependent protein kinases (CDPKs) are implicated in parasite invasion processes.
Purpose of the Study:
- To investigate the role of calcium-dependent protein kinase 4 (EtCDPK4) in E. tenella invasion of host cells.
- To characterize the expression, activity, and functional domains of EtCDPK4.
Main Methods:
- Identification and characterization of EtCDPK4 cDNA.
- Quantitative real-time PCR and Western blotting to assess expression levels.
- In vitro invasion inhibition assays using antibodies and specific inhibitors.
- Indirect immunofluorescence assays for protein localization.
- In vitro kinase activity assays and inhibitor screening.
Main Results:
- EtCDPK4 expression was higher in sporozoites, with greater translation in second-generation merozoites.
- Antibodies against EtCDPK4 inhibited E. tenella sporozoite invasion by approximately 52%.
- Recombinant EtCDPK4 (rEtCDPK4) demonstrated Ca2+-dependent protein kinase activity.
- Specific inhibitors (W-7, H-7, H-89, myristoylated peptide) significantly reduced parasite invasion.
Conclusions:
- EtCDPK4 plays a significant role in the invasion of chicken cecal epithelial cells by E. tenella.
- EtCDPK4 is a Ca2+-dependent protein kinase essential for parasite infectivity.
- Targeting EtCDPK4 activity presents a potential strategy for controlling Eimeria infections.
