The mechanism of Jurkat cells apoptosis induced by Aggregatibacter actinomycetemcomitans cytolethal distending toxin
- 313 Downloads
Cytolethal distending toxin (CDT) which is produced by Aggregatibacter actinomycetemcomitans causes apoptosis in lymphocytes. But the specific mechanism is not clear. The aim of our research was to investigate the effect and mechanism during this process. The wild-type CdtA, CdtB, CdtC (CdtAW, CdtBW, CdtCW) and mutant CdtB (CdtBM) were expressed and purified respectively and the purity of each subunit was examined by BandScan software. And the type I deoxyribonuclease and PI-3,4,5-triphosphate (PI-3,4,5-P3, PIP3) phosphatase activity were detected by DNA agarose gel electrophoresis and enzyme-linked immunosorbent assay respectively. The cell apoptosis rates were analyzed by flow cytometry. The morphological changes of apoptosis cells were observed by confocal laser scanning microscopy. The protein expression of Bax and Bcl-2 was examined by western blot. Differentially expressed apoptosis-related proteins were identified based on isobaric tags for relative and absolute quantitation technology. In the present study we found that: (i) recombinant wild-type CdtA, CdtB and CdtC (CdtAW, CdtBW, CdtCW) and mutant CdtB (CdtBM) were correctly expressed and the purity of each protein was higher than 80%, (ii) the average apoptosis rate in wild-type CDT (CDTW) treated groups was 50.54%, which was significantly higher than the controls (4.71%) and mutant CDT (CDTM) treated groups (5.58%) (p < 0.05), (iii) morphological changes of apoptosis were observed in CDTW treated cells, (iv) the expression of Bax protein was significantly increased in CDTW treated cells, while Bcl-2 protein expression was significantly decreased, (v) 17 apoptosis-related proteins were expressed differentially, among which 10 proteins (SMNDC1, TNFRSF10B, UBE2I, ITM2A, CASP3, P53, EIF1, TCF3, HMGN5, CASP8) were up-regulated and 7 proteins (RRM2, TPX2, KIF11, NUCKS1, TOP2A, XRCC1, PTPLAD1, RRM2) were down-regulated, (vi) one possible apoptotic pathway [Ubc9 (UBE2I)/P53/DR5 (TNFRSF10B)/Caspase-8 (CASP8)/ Caspase-3 (CASP3)] was selected and partially proved.
KeywordsAggregatibacter actinomycetemcomitans Cytolethal distending toxin Jurkat cells Apoptosis Proteomics Signal transduction
This study was supported by grants from the National Natural Science Foundation of China (Grant No. 81170962,81470749), Natural Science Research of University of Jiangsu Province (Grant No. 14KJD320001) and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, 2014-37). We also thank American Journal Experts for revising the English used in this article.
Compliance with ethical standards
Conflict of interest
There are no conflicts of interest to declare.
- 1.Shenker BJ, Dlakic M, Walker LP, Besack D, Jaffe E, LaBelle E, Boesze-Battaglia K (2007) A novel mode of action for a microbial-derived immunotoxin: the cytolethal distending toxin subunit B exhibits phosphatidylinositol 3,4,5-triphosphate phosphatase activity. J Immunol 178:5099–5108CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Rompikuntal PK, Thay B, Khan MK, Alanko J, Penttinen AM, Asikainen S, Wai SN, Oscarsson J (2012) Perinuclear localization of internalized outer membrane vesicles carrying active cytolethal distending toxin from Aggregatibacter actinomycetemcomitans. Infect Immun 80:31–42. doi: 10.1128/IAI.06069-11 CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Jankova L, Chan C, Fung CL, Song X, Kwun SY, Cowley MJ, Kaplan W, Dent OF, Bokey EL, Chapuis PH, Baker MS, Robertson GR, Clarke SJ, Molloy MP (2011) Proteomic comparison of colorectal tumours and non-neoplastic mucosa from paired patient samples using iTRAQ mass spectrometry. Mol Biosyst 7:2997–3005. doi: 10.1039/c1mb05236e CrossRefPubMedGoogle Scholar
- 10.Kang J, de Brito Bezerra B, Pacios S, Andriankaja O, Li Y, Tsiagbe V, Schreiner H, Fine DH, Graves DT (2012) Aggregatibacter actinomycetemcomitans infection enhances apoptosis in vivo through a caspase-3-dependent mechanism in experimental periodontitis. Infect Immun 80:2247–2256. doi: 10.1128/IAI.06371-11 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Shima H, Suzuki H, Sun J, Kono K, Shi L, Kinomura A, Ikura T, Ikura M, Kanaar R, Igarashi K, Saitoh H, Kurumizaka H, Tashiro S (2013) Activation of the SUMO modification system is required for the accumulation of RAD51 at sites of DNA damage. J Cell Sci 126: 5284–5292. doi: 10.1242/jcs.133744 CrossRefPubMedGoogle Scholar
- 39.Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, Gottlieb E, Green DR, Hengartner MO, Kepp O, Knight RA, Kumar S, Lipton SA, Lu X, Madeo F, Malorni W, Mehlen P, Nuñez G, Peter ME, Piacentini M, Rubinsztein DC, Shi Y, Simon HU, Vandenabeele P, White E, Yuan J, Zhivotovsky B, Melino G, Kroemer G (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19:107–120. doi: 10.1038/cdd.2011.96 CrossRefPubMedGoogle Scholar