The Function And Mechanism Of ROP5 And ROP16 In Neospora Caninum

Neospora caninum(N.caninum),of the Apicomplexa phylum,is a common cause of abortions in cattle and nervous system dysfunction in dogs.Compared to T.gondii and Plasmodium,the researches on N.caninum have few reports about its pathogenic mechanism.The study on a few.caninum rhoptry proteins' functions and mechanisms were scheduled to develop.Many kinds of rhoptry proteins(ROPs)of Apicomplexa have been reported and some of them play an important role in the lives of parasites.Some rhoptry proteins have been identified as virulence factors in T.gondii and Plasmodium.At the moment,rhoptry proteins in N.caninum are reported lesser and ROP18 has been proved a pseudogene in our previous research.The main research object of the thesis is ROP5 and ROP16 because of normal expression of them.Bioinformatic analyses indicated that N.caninum may have 3 copies of ROP5 gene.We successfully constructed ROP5 gene knockout strains(AROP5)using homologous recombination method and ?ROP5 complementary strain(AROP5-cm).According to a study of ROP5 gene-regulating and original strains,AROP5 had less plaque space,weakened invasion capacity and slower intracellular growth.The results showed significantly lower cerebral load of ?ROP5 after intraperitoneal injected BALB/c mice with 1�106 tachyzoites than the load of the Ncl strain,as well as a loss of pathogenicity for the?ROP5 with 3�106 parasites.All this corroborated ROP5 is a key factor associated with N.caninum pathogenicity for mice.The studies comparing ?ROP5,AROP5-cm and Ncl showed decreased GRA7 and increased ROP17 transcription in ?ROP5.Meanwhile,GRA7 couldn't correctly locate on the parasitophorous vacuole membrane(PVM)and ROP17 was wrongly diffusely distributed in the cytoplasm.All parasites could stimulate the host cell autoimmune response,and generate GBP1,GBP2,IRGb6 and IRGd induced by IFN-y,but the expression of GBP1,GBP2 and IRGb6 acted by?ROP5 were lower than other strains.The recruitment of GBP1 and GBP2 was significantly enhanced about 40%on the ?ROP5 PVM,and the result implied ?ROP5 were more easily killed.Above evidences initiatively proved that the escape of N.caninum from immnue mediated by ROP5 regulating the expression of GBP1 and GBP2.Based on the previous research,N.caninum Ncl could phosphorylate STAT3Y705,but the mechanism is not clear.Bioinformatic analyses showed that ROP16 has a conserved nuclear localization signal(NLS)and a phosphate kinase domain(PKc),and STAT3 is a downstream gene of ROP16 by KEGG pathway analysis.To examine the relation between ROP16 and phosphorylating STAT3,we still used the homologous recombination method and constructed ROP16 gene-knockout strains(?ROP16),the complement strains(AROP16-cm)and the overexpression strains(ROP16-oe).According to the observed parasites phenotypes,?ROP16 had smaller plaques and slower intracellular growth compared to Ncl,in contrast to ?ROP16-cm and ROP16-oe.The results showed significantly lower cerebral load of ?ROP16 after intraperitoneal injected BALB/c mice with 1�106 tachyzoites and higher of ?ROP16-cm and ROP16-oe than the load of Ncl group,as well as attenuate pathogenicity for the ?ROP16 and virulence for AROP16-cm and ROP16-oe with 3�106 parasites.That suggested that ROP16 plays an important role for N.caninum pathogenicity.In addtion,the deletion of ROP16 declined the host cell STAT3 phosphorylation level,and ROP16 could phosphorylate STAT3 in HEK293T cells by transfection.That indicated N.caninum ROP16 taked part in the activation of STAT3 signaling pathway in host cells.The results of research on mutROP16NLS and mutROP16PKc showed that ROP16 could enter the host nucleus depending on NLS and continuously phosphorylate STAT3 depending on PKc.We detected macrophages ANA1 apoptosis induced by ROP16 gene-regulating strains and Ncl,and the apoptosis was lower in AROP16 group than other groups.The result showed that N.caninum ROP16 induced the host cell apoptosis.