A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis

Abstract

Screening the anticoccidial drug targets is very important for developing novel drugs and revealing the molecular basis of drug resistance in coccidia. Due to high effectivity and safety, diclazuril was used widely in the poultry industry. To assess the roles of the serine/threonine protein phosphatase type 5 of second-generation merozoites in Eimeria tenella (EtPP5) in the anticoccidial activity of diclazuril against chicken coccidiosis, EtPP5 was cloned using reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Ultrastructural changes in second-generation merozoites and mRNA expression level of EtPP5 were monitored by transmission electron microscopy (TEM) and quantitative real-time PCR, respectively. The results showed that the full length of the cloned EtPP5 cDNA (2,495 bp) encompassed a 1,647-bp open reading frame encoding a polypeptide of 548 residues with an estimated molecular mass of 60.82 kDa and a theoretical isoelectric point of 5.89. Molecular analysis of EtPP5 reveals the presence of a C-terminal phosphatase domain and an extended N-terminal tetratricopeptide repeat motif, a typical feature of protein phosphatases. The cDNA sequence has been submitted to the GenBank database with accession number JX987508. EtPP5 shared 89% homology with the published sequence of a PP5 ortholog of Toxoplasma gondii at the amino acid level (GenBank XP_002364442.1). TEM observed that diclazuril induced ultrastructural changes in second-generation merozoites. Quantitative real-time PCR analysis showed that compared with the control group, the level of EtPP5 mRNA expression was significantly downregulated by 51.4% by diclazuril treatment. The high similarity of EtPP5 to previously described PP5 of other organisms, as well as its downregulated expression and connection with apoptosis in the second-generation merozoites induced by diclazuril, suggests that it could act an important role in understanding the signaling mechanism underlining the diclazuril-induced merozoites apoptosis.

Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

“…In mammals, PP5 regulates intracellular biological activities as diverse as cellular proliferation, differentiation, migration, survival and death, and DNA damage repair [8]. A recent study revealed that PP5 has an important role in the signaling mechanism associated with the diclazuril-induced merozoite apoptosis in Eimeria tenella [9]. To date, our knowledge of insect PP5 is still limited.…”

Diclazuril Protects against Maternal Gastrointestinal Syndrome and Congenital Toxoplasmosis

“…The fact that mammalian systems lack the protochlorophyllidae related diclazuril/herbicide binding region results in a very high therapeutic index for diclazuril and related agents. In addition, diclazuril is shown to down-regulate expression of serine/threonine protein phosphatase (EtRACK) in second-generation merozoite of Eimeria tenella to induce apoptosis [31]. EtRACK has about 98% homology with Toxoplasma that demonstrates a possible mechanism of action for diclazuril.…”

Novel Synergistic Protective Efficacy of Atovaquone and Diclazuril on Fetal-Maternal Toxoplasmosis

“…Therefore, diclazuril binds the chloroplast epitopes to interact with the D1 protein of the photosynthetic reaction hub of Toxoplasma organelles, without any harmful effect on the host cells. Additionally, diclazuril can downregulate expression of serine/threonine protein phosphatase (EtRACK) in merozoites of Eimeria tenella chicken coccidiosis which results in apoptosis [46]. EtRACK has about 98% homology with Toxoplasma organisms which demonstrates a predicted mechanism of action for diclazuril against toxoplasmosis.…”