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| Biological Function of Cytolethal Distending Toxin (CDT) of Haemophilus parasuis |
| 1,袁 秀芳2, 1, 1 |
1.
2. 浙江省农业科学院
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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.
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Received: 27 April 2015
Published: 23 November 2015
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