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.
Highlights
The protozoan phylum Apicomplexa comprises thousands of obligate intracellular parasites, many of which cause significant human and animal health problems
The full-length EtCDPK4 cDNA was 2499 bp with a single open reading frame (ORF) of 1803 bp encoding a polypeptide of 600 amino acid residues with a calculated molecular mass of approximately 68.3 kDa
TMPRED analysis of the transmembrane regions of the EtCDPK4 amino acid sequence found a transmembrane region in the Val302—Ile324 position
Summary
The protozoan phylum Apicomplexa comprises thousands of obligate intracellular parasites, many of which cause significant human and animal health problems. The invasion of host gut epithelial cells by Eimeria species is a complex, multistep process that begins with the apical attachment of the parasite to the host cell. This is followed by rapid internalization to form an intracellular, parasitophorous vacuole (PV) that encloses the newly invaded parasite, enabling its survival within the host [6]. Eimeria need to egress from infected cells and reinvade uninfected cells In response to these events, parasites have developed regulatory mechanisms for self-proliferation and invasion. Secretion by these organelles is controlled by intracellular calcium as a second messenger, which is important in signal transduction cascades, including for protein secretion, gliding motility, invasion of and egress from host cells, proliferation and differentiation [9]
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