Abstract：Haemophilus parasuis is a common bacterial agent of porcine (Susscrofa domestica), and the infection of this pathogen is characterized by fibrinous to fibrinopurulent polyserositis and polyarthritis. Cytolethal distending toxin (CDT) is proposed as an important virulent factor of H. parasuis, but some biological and immunological characteristics of CDT are still to be studied. Highly purified CDT holotoxin was prepared through co-refolding of 3 subunits by dialysis at 4 ℃ into a native buffer and purification by Ni-NTA affinity chromatography and gel-filtration chromatography. Cytotoxicity of CDT on both Marc145 and porcine peripheral blood lymphocyte (PBLC) was studied. Moreover, neutralization effect of specific antibody of CDT subunits against its cytotoxicity was measured. It showed that stable ternary complex were formed following co-refolding with 3 recombinant CDT subunits, and the formation of triplex CDT was confirmed by immunoprecipitation assay with purified IgG against CdtC subunit. The CDT holotoxin could induce cell distention by 2~4 fold of the original size and nuclear enlargement on Marc145, while any one or 2 of the 3 subunits could not induce typical cytotoxicity. Both CdtB and CdtC anti-serum could inhibit cytotoxicity of the assembled CDT holotoxin, and CdtC anti serum showed the best neutralization effect with the titer of 1∶16, while no neutralization effect of CdtA anti-serum was observed. The unassembled recombinant subunits mixture was significantly more susceptible to the neutralization effect of both CdtB and CdtC anti-serum than the assembled CDT holotoxin (P＜0.05). Furthermore, H. parasuis CDT blocked proliferation of mitogen activated PBLC, and the proliferation of PBLC was significantly suppressed when applied 20 or 10 μL holotoxin to ConA or phorbol-My-ryslate-acetate (PMA) activated cells, respectively (P＜0.05). When cultured in vitro, virulent and non-virulent H. parasuis secreted proteins showed the same cytotoxicity effect with the titer of 1∶512; The result of immunoprecipitation assay with purified IgG against CdtC subunit showed that CDT in the secreted proteins was present as ternary complex. Hence, H. parasuis CDT recombinant subunits could form holotoxin in vitro, and the assembled holotoxin exhibited effective bio-activity; Humoral immune responses against CdtB and CdtC could completely block the cytotoxicity caused by the holotoxin; Holotoxin showed significant proliferation suppression effect on mitogen activated PBLC cells; H. parasuis secreted proteins of 2 strains with great variation in virulence showed the same cytotoxicity, and CDT in the secreted proteins was presented as triplex holotoxin. H. parasuis CDT exhibited potent immunosuppressive effect, and it was possible to play important roles in colonization and causing systemic infection in the host, while it was not responsible for the extensive virulence variation between strains. This study provides novel information for understanding the pathogenesis of H. parasuis.
[1]曹会敏, 李军星, 袁秀芳, 等.副猪嗜血杆菌毒力与非毒力菌株菌体蛋白的比较蛋白质组学分析[J].农业生物技术学报, 2013, 21(7):870-878
[2]陈西, 王湘如, 徐晓娟, 等.副猪嗜血杆菌细胞致死膨胀毒素的细胞毒性研究[J].畜牧兽医学报, 2011, 42(12):1750-1755
[3]Costa-Hurtado M, Aragon V.Advances in the quest for virulence factors of Haemophilus parasuis[J].The Veterinary Journal, 2013, 0(198):571-576
[4]Jinadasa R N, Bloom S E, Weiss R S, et al.Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression,leading to apoptosis of a broad range of mammalian cell lineages[J].Microbiology, 2011, 0(157):1851-1875
[5]Johnson W M, Lior H.Response of Chinese hamster ovary cells to a cytolethal distending toxin (CDT) of Escherichia coli and possible misinterpretation as heat-labile (LT) enterotoxin[J].FEMS Microbiology Letter, 1987, 0(43):19-23
[6]李军星, 王一成, 袁秀芳, 等.副猪嗜血杆菌亚基原核表达及其抗原性鉴定[J].中国预防兽医学报, 2012, 34(9):746-748
[7]李艳玲，李建军，周泷, 等.副猪嗜血杆菌细胞致死膨胀毒素亚基的克隆表达及细胞毒性分析[J].中国预防兽医学报, 2013, 35(6):449-452
[8]Nesic D, Hsu Y, Stebbins C E.Assembly and function of a bacterial genotoxin[J].Nature, 2004, 0(429):429-433
[9]Oliveira S, Pijoan C.Haemophilus parasuis: new trends on diagnosis,epidemiology and control[J].Veterinary Microbiology, 2004, 0(99):1-12
[10]Shenker B J, Hoffmaster R H, Zekavat A, et al.Induction of apoptosis in human T cells by Actinobacillus actinomycetemcomitans cytolethal distending toxin is a consequence of G2 arrest of the cell cycle[J].Journal of Immunology, 2001, 0(167):435-441
[11]Thelestam M, Frisan T.Cytolethal distending toxins[J].Reviews of Physiology Biochemistry and Pharmacology, 2004, 0(152):111-133
[12]Ueno Y, Ohara M, Kawamoto T, et al.Biogenesis of the Actinobacillus actinomycetemcomitans cytolethal distending toxin holotoxin[J].Infection and Immunity, 2006, 0(74):3480-3487
[13]Yue M, Yang F, Yang J, et al.Complete Genome Sequence of Haemophilus parasuis SH0165[J].Journal of Bacteriology, 2009, 191(4):1359-1360
[14]Zhang B, He Y B, Xu C G, et al.Cytolethal distending toxin (CDT) of the Haemophilus parasuis SC096 strain contributes to serum resistance and adherence to and invasion of PK-15 and PUVEC cells[J].Veterinary Microbiology, 2012, 157(1-2):237-242
[15]Zhou M G, Zhang Q, Zhao J P, et al.Haemophilus parasuis Encodes Two Functional Cytolethal Distending Toxins: CdtC Contains an Atypical Cholesterol RecognitionInteraction Region[J].PLoS ONE, 2012, 7(3):e32580-e32580
