The Effect And Mechanism Research Of Theileria Annulata Infection On Host Cells Telomere Length And Telomerase Activity

Theileria annulata is a blood protozoan parasite which transmited by the genus of Hyalomma ticks.As an intracellular protozoan parasite,Theileria annulata could infect bovine B cells,machrophage cells,dendritic cells and red blood cells.After sporozites invaded in immune cells,T.annulata develop into schizont stage.The host cells were then transformed by T.annulata and obtained unlimited proliferate ability that as tumor cells.Up untill now,many signal pathways of host cells were confirmed could be affected by T.annulata infection,but there still have many questions that are needed to illustrate for cells transformation caused by Theileria.Telomerase,a protein-RNA complex,is plays an important role to maintain telomere length and biological activities of cells.And the activity of telomerase is directly related with the protein expression level of telomerase reverse transcriptase(TERT).Telomerase usually is inactivated in somatic cells,but reactivated in more than90% tumor cells.Whether telomerase is activated or not in T.annulata transoformed cells and the activation mechanism has not reported yet.Aim to understand the effect of T.annulata on cells phenotype,the effects and mechanism of T.annulata infection on host cells telomere length and telomerase activity.In the present study,T.annulata transformed bovine dendritic cells was established through in vitro infection and cells phenotype and antigen endocytosis was analyzed;analyzed the change of genes transcription level between the normal and infected cells;the change of telomere length,telomerase activity and relative genes expression was analyzed in three types of T.annulata infected cells;screened and identified parasites protein interacting with b TERT;and to determinate the effect of parasite protein on b TERT expression and telomerase activity of host cells.The results of the present study will provide some new data for the studies on cell transformation by T.annulata.The main research results are as follows:1.Establishment of T.annulata transformed DC cell line and analysis a part of phenotyps and gene transcription levels: Succesfully established T.annulata transformed bovine DCs(Ta DC).After infection,the DC cells can’t express CD14 anymore and the expression of CD11 c,CD11b and CD86 was significantly down regulated.And the expression of CD205,MHC Ⅱ and CD45 was significantly up regulated.The endocytosis function of Mo DC was inhibited by the infection of T.annulata.From the transcriptome data,2027 host genes were up regulated and those genes involved in many signal pathways including PI3K-AKT、MAPK、Fox O、JAK-STAT and TNF,etc.Most importantly,the TERT gene,which plays an important role in cancer cells,was found significantly up regulated in T.annulata infected cells.2.The effect of T.annulata infection on telomere length and telomerase activaty of host cells: Compared with normal somatic cells,in three types of T.annulata transformed cells Ta NM1,Ta BC and Ta DC,telomere length and telomerase activity was significantly up regulated,and the transcription level of b TERT and b TERC was also up regulated.But these changes will be disappeared after the elimination of the parasites.When the telomerase activity was inhibited by its specific inhibitor,the cells stopped grow and turn to apoptosis,which indicating telomerase play an important role in T.annulata transformed cells.In addition,inhibition of b HSP90 confirmed that T.annulata affects telomerase activity might through the b HSP9 0-AKT-b TERT protein complex.3.Screenning of T.annulata proteins interact with bTERT: Constructed T.annulata macroschizont c DNA library and recombinant b TERT bait plasmid.7 parasite proteins which are possible interacting with b TERT,were screened out by using Y2 H system.Parasite proteins Dna J and ETIF2 were confirmed could interacts with b TERT by Co-IP and Bi FC assays.4.Mechanism of the interaction between ETIF2 and bTERT: With the antibody against Dna J and ETIF2 protein which derived from mouse,confocal microscopic results shown that Dna J protein mainly locates on the surface of macroschizont and ETIF2 protein distributes on the surface of macroschizont and in the cytoplasm of host cells.Constructed the recombinant Dna J/ETIF2-Plvx-IRES plasmids and established TaNM1 cells which express those proteins.The results shown that both Dna J and ETIF2 protein could enhances the transcription level of b TERT and b TERC,but only ETIF2 protein was able to up regulate b TERT protein expression level and telomerase activity.After expressed the different domain of ETIF2 and b TERT,Co-IP results confirmed that the e IF5/e IF2 B binding domain of ETIF2 interacts with the RT domain of b TERT,which confirmed the reaction site between ETIF2 and b TERT protein.In conclusion,the present study demonstrated that T.annulata infection could active telomerase and to extend telomere lenght of host cell for first time.And T.annulata could bind to host cells b TERT via parasite protein ETIF2 to activate telomerase and maintain the contiuous proliferation of host cells.The results of this study provide new data for further elucidating the mechanism of cells transformation induced by T.annulata.