Functional Study Of Atypical Kinase RIO2 In Schistosoma Japonicum

As a dioecious genus of trematode,schistosomes can infect many animal species thus leading to severe parasitic disease that has an impact on more than 230 million people worldwide. Schistosoma japonicum is prevalent in southern part of China, and the main drug for schistosomiasis treatment is praziquantel. However,due to the absence of valid vaccine and possible emergence of drug-resistance worm strain,there is a long way to go to fight against schistosomiasis.Protein kinases can phosphorylate its substrate and change the substrate’s conformation or/and activity and thus to regulate cellular process, this particular character makes them ideal targets for drug design. Protein kinases can be classified into two groups: eukaryotic protein kinases(e PKs) and atypical protein kinases(a PKs). Different from e PKs,a PKs do not have conserved catalytic structural domain,but the kinase activity remains. RIO kinase is among the 13 families of a PKs, and is conserved in almost all species. To date, four members of RIO kinase family have been identified: Rio1, Rio2, Rio3 and Rio B, which only exists in archaea. An study on Saccharomyces cerevisiae unveiled that Rio kinases play important roles in pre-ribosome assembly and cell cycles, and Rio2 is essential in 18 S r RNA processing and metaphase-anaphase transition in mitosis. Above all, no light has been shed on the function of RIO kinases in multi-cellular parasite, not to mention in Schistosoma japonicum. Therefore, we pursued our study on the function of Rio2 kinase in Schistosoma japonicum on the basis of our former work.1)RNA interference(RNAi) of Schistosoma japonicum Rio2 and Plk1 Because Rio2 was discovered as a novel substrate of polo-like kinase 1(Plk1), we interferred these two genes with double-strand RNA(ds RNA) for 9 days with worms collected 28 days post infection. The efficiency of RNAi experiment is 75-95%.2)Phenotype observation after RNAi We took measurements to determine phenotype change: the eggs laid by female worms during 3-5 days, 5-7 days, 7-9 days in interference phase were counted manually, but no significant difference was detected among groups; the length of interfered worms were measured under microscope, but no significant difference was detected among groups. This suggests that interference of Rio2 and Plk1 in this study has no impact on the egg-laying and worm development. By adding Ed U into culture medium, cells in mitosis was labeled, collecting samples after cultivation and observed with CLSM, we found out that: when knocking down Plk1 only, the mitosis process was significantly suppressed in reproductive organs of Schistosoma japonicum; but the mitosis process was slightly suppressed when knocking down Rio2; and the double-knock down group reveal a similar result with Plk1 knock down group. This suggests that Plk1 overpowered Rio2 in regulating the mitosis phase. By staining the worms with carmine and observing with CLSM, we found out that: when knocking down Plk1 only, the number of immature cells in ovary and vitelline gland was reduced; but the number of cells in vitelline duct and eggs in uterus increased a lot when knocking down Rio2; and the double-knock down group reveal a similar result with control group. This suggests that Plk1 and Rio2 may compete with each other functionally.3)Transcriptional level of apoptotic related genes after RNAi We collected female worms after RNAi experiment and extracted RNA for real-time PCR, we detected the transcriptional level of three apoptotic genes: Caspase3, Caspase7 and CIAP1, the result indicates that knocking down Rio2 or Plk1 does not lead to significant change in the transcriptional levels of these three apoptotic genes.4) Preparation and application of the egg-yolk Ig Y of Schistosoma japonicum Rio2 After expression and purification of truncated Rio2 recombinant protein, white Leghorns were immunized, and egg-yolk Ig Y was extracted from eggs laid by these chickens, ELISA showed that the titer of the antibody was 3200. The activity of immune response was identified by Western blot with both recombinant protein and Schistosoma japonicum protein. The results indicated that the egg-yolk may bind specifically with the recombinant protein but not with the Schistosoma japonicum protein.This study knocked down Rio2 and Plk1 genes successfully in Schistosoma japonicum; analyzed the phenotype change after RNAi, including mitosis, morphology of reproductive organs, egg number, worm length, and detected the transcriptional level of apoptotic genes after RNAi; prepared egg-yolk Ig Y against Rio2 protein. These findings set foundation for further investigating of the function of RIO kinases in Schistosoma japonicum.