Preliminary Mechanism Of Jurkat Cells Apoptosis Induced By Aggregatibacter Actinomycetemcomitans Cytolethal Distending Toxin

Objective:To investigate the preliminary mechanism of Jurkat cells apoptosis induced by Aggregatibacter actinomycetemcomitans(A.actinomycetemcomitans)cytolethal distending toxin(CDT)so as to find out the potential mechanisms involved in this process.Methods:The wild-type cdtA,cdtB,cdtC(cdtA~W,cdtB~W,cdtC~W)and mutant cdtB(cdtB~M)were constructed by pET-15b plasmid,then the protein expression was induced by IPTG,examined by SDS-PAGE gels and stained with coomassie blue staining(CBS)R250 solution.The recombinant proteins were purified through pre-installed Ni-HisTrap HP column respectively and the purity of each subunit was examined by BandScan software.The Deoxyribonuclease I(DNase I)and PI-3,4,5-triphosphate(PIP3)phosphatase activity of CdtB subunit were detected by DNA agarose gel electrophoresis and ELISA method respectively.The cell apoptosis rates were analyzed by flow cytometry(FCM).The morphological changes of apoptosis were observed by transmission electron microscope(TEM)and confocal laser scanning microscopy(CLSM).The protein expressions of Bax and Bcl-2 were examined by Western blot.Differientially expressed apoptosis-related proteins were identified based on iTRAQ technology.One possible pathway was selected and partially verified by Western blot.Results:Recombinant CdtA~W,CdtB~W and CdtC~W and CdtB~M protein were correctly expressed and the purity reached to 80%.The CdtB~W could enzyme the pET-32a DNA and owned the PIP3 activity.The average apoptosis rate in CDT~W treated groups was 50.54%,much higher than the controls(4.71%)and CDT~M treated groups(5.58%)(p<0.05).Morphological changes of apoptosis were observed in CDT~W treated cells.The expression of Bax protein was significantly increased in CDT~W treated cells,while that of Bcl-2 protein was significantly decreased.Seventeen apoptosis-related proteins expressed differientially,among which ten proteins(SMNDC1?TNFRSF10B?UBE2I?ITM2A?CASP3?P53?EIF1?TCF3?HMGN5?CASP8)were up-regulated and seven proteins(RRM2?TPX2?KIF11?NUCKS1?TOP2A?XRCC1?PTPLAD1?RRM2)were down-regulated.Finally,one possible apoptosis pathway[Ubc9(UBE2I)/P53/DR5(TNFRSF10B)/Caspase-8(CASP8)/Caspase-3(CASP3)]was selected and partially proved.Conclusion:CdtA~W,CdtB~W,CdtC~W,CdtB~M,and the CDT holotoxin with biological activity were successfully acquired.CdtB~W owned both DNase I and PIP3 activities,and CDT~W holotoxin could induce significant apoptosis of Jurkat cells.Furthermore,the key apoptosis-related proteins involved in the process were successfully detected.Such preliminary findings would contribute to a further understanding of Jurkat cells apoptosis induced by A.actinomycetemcomitans CDT and could be helpful to the diagnosis and treatement of localized aggressive periodontitis(LAgP).