Mechanism of diclazuril acting on actin depolymerizing factor against Eimeria tenella

Eimeria tenella (E. tenella), an obligate intracellular apicomplexan parasite, invades chicken cecal epithelial cells, causing severe chicken coccidiosis. During the host invasion process, actin provides the driving force for E. tenella. Actin depolymerization factor (ADF), an actin binding protein, can regulate actin filaments turnover and cytoskeleton reconstruction, playing a critical role in parasite gliding invasion of host cells. Diclazuril, a benzeneacetonitrile anticoccidial agent, exhibits potent activity against E. tenella. However, mechanism of diclazuril action on ADF against E. tenella remains unclear. In this study, a chicken model infected with E. tenella was established, and the second-generation merozoites were subsequently harvested from both infected and diclazuril-treated groups. EtADF subcellular localization was assessed by immunofluorescence, while total and phosphorylated EtADF (p-EtADF) expression levels were quantified via western blotting. Subsequently, the in vitro depolymerization and polymerization kinetics of actin were monitored using a fluorescence spectrophotometer. Actin co-sedimentation assay and SDS-PAGE were employed to assess the biological activity of EtADF, while the F-actin morphology was examined using transmission electron microscopy (TEM). Results demonstrated that EtADF was localized in cytoplasm of E. tenella second-generation merozoites, and diclazuril reduced total EtADF expression while increasing p-EtADF levels. Actin dynamics assays, co-sedimentation analysis, and TEM revealed that EtADF exhibits F-actin binding, bundling and depolymerization activities, as well as G-actin sequestering and polymerization inhibition. Diclazuril effectively inhibited these activities of EtADF. Collectively, these findings provide important insights into molecular mechanism underlying the anticoccidial effect of diclazuril, and highlight EtADF as a potential novel drug target.