Molecular characterization and analysis of the drug resistance-associated protein phosphoglycerate kinase of Eimeria tenella.

Abstract

Coccidiosis is a parasitic disease caused by Eimeria spp., and the emergence of drug resistance has seriously affected the control of the disease. Using RNA-seq, we previously found that phosphoglycerate kinase of Eimeria tenella (EtPGK) was differentially downregulated in diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains compared with drug-sensitive (DS) strain. In this study, we further analysed the characteristics and functions of EtPGK to find the possible mechanism of drug resistance of E. tenella. Quantitative real-time PCR (qRT-PCR) and western blot found that EtPGK was highly expressed in sporulated oocysts, followed by sporozoites and second-generation merozoites of E. tenella. Indirect immunofluorescence localization showed that EtPGK was located mainly in the cytoplasm and on the surface of the parasites. Invasion inhibition assays showed that anti-rEtPGK antibody significantly inhibited the invasion of parasites. Further studies using qRT-PCR and western blot found that the transcription and translation levels of EtPGK were downregulated in both resistant (DZR and MRR) strains compared with the DS strain, and the transcription level correlated negatively with the drug concentration. The enzyme activity assay revealed that EtPGK enzyme activity was decreased in the DZR strain compared with the DS strain. qRT-PCR revealed that the mRNA transcription level of EtPGK was significantly downregulated in the field DZR strain and salinomycin-resistant strain compared with the DS strain. These results suggested that EtPGK has other important roles that are separate and distinct from its function in glycolysis, and it might be involved in the development of drug resistance of E. tenella.