Cloning And Characterization Of EmAurora From Echinococcus Multilocularis

Alveolar echinococcosis(AE)is a zoonotic parasitic disease caused by the metacestode larvae of Echinococcus multilocularis,which is considered to be the most lethal helminthic disease.The germinative cells are the basis of the growth and development of the larvae of E.multilocularis,and are the only cells which can proliferate.Cell mitosis is a strictly regulatory and highly ordered process.Aurora kinasesare important regulators of mitosis.They are involved in the maturation of the centrosome,the establishment of the spindle and the normal separation of chromosome and cytoplasm.By searching the genome database of E.multilocularis,we found that there were two Aurora kinase homologous,which were named EmAurora A and EmAurora B.The full-length coding sequences ofE.mwultilocularis Aurorahomologouswere amplified by molecular cloning technology,and the gene structure,functional domain,and protein tertiary structure were analyzed.The results showed that the amino acid sequences of EmAurora A and EmAurora B were highly conserved and that they exibited similar tertiary structure with mammalian Aurora A and Aurora B,respectively.The protein and mRNA expression of EmAurora in the larvae of E.multilocularis were detected by Western blot and RT-PCR.The results showed that EmAurora are constitutively expressed in the protoscoleces and vesicles.The wild type EmAurora A and EmAurora B were recombined into pcDNA3.3-HA vectors and the mutant were constructed in which the threonine(T)in the kinase domain RxT motif was mutated into alanine(A)and were transfected into 293T cells respectly,Cell proteins were extracted and the target proteins were obtained using Immunoprecipitation.The effect of EmAurora on phosphorylation of histone H3 was analyzed by the vitro kinase assay.The result showed that wild-type kinase could phosphorylate histone H3 while mutant kinase had almost no ability to phosphorylate H3,which indicated that the kinases could hadphosphorylate substrate histone H3and the RxT motif was required for kinase functions.Subsequently,the Aurora inhibitors VX680,MLN8024,AZD1152 were used to treat the metacestode larvae of E.multilocularis.The results showed that Aurora inhibitors did not significantly affect the survival of the vesicles andprotoscoleces,but it could significantly inhibit the growth of the vesicle and the development of the protoscoleces.Further analysis by EdU and DAPI stainning revealed showed that Aurora's inhibitors could effectively inhibit the proliferation ofE.multilocularis germinative cells and could also result in polyploidy cells.These results suggest that EmAurora A and EmAurora B are involved in the mitotic process of germinative cells of E.multilocularis,and are potencial new drug targets for the treatment of alveolar echinococcosis.