| Haemophilus parasuis(H. parasuis) is the pathogenic agent of Glasser’s disease of pigs, which presents clinically with fibrinous polyserositis, polyarthritis and meningitis. Cytolethal distending toxin(CDT) is an important virulence factor of H. parasuis. It is composed of 3 subunits: CdtA, CdtB and CdtC. To investigate the cytotoxicity that H. parasuis CDT(HpCDT) exposed to the host cells produced, we used the PAM and PK-15 cells as a model to study the cytotoxicity via the flow cytometry, laser confocal microscope, western blot and real-time PCR.The signal peptides of 3 subunits were predicted by Predsi, Signal-Blast and Signal-3L, the results showed that the N-terminal 1-19 aa, 1-21 aa and 1-19 aa were the signal of CdtA, CdtB and CdtC, respectively, and truncated the signal peptides, all 3 subunits successfully expressed in soluble form. Purified CdtB could cleaved circular and linear DNA, which displayed that CdtB had DNase activity in vitro. Meanwhile, Purified CdtB was able to induce the PK-15 cells producing γ-H2 A.X, which showed CdtB had DNase activity in vivo and caused DNA double strand damage. Flow cytometry analysis showed CdtB alone could induce cell G2/M arrest and cell apoptosis, which would be enhanced by CdtA or/and CdtC. Further study showed CDT holotoxin could lead to significant cell distension, G2 arrest and apoptotic death. In addition, using pifithrin-α, the apoptosis induced by CDT was significantly inhibited. cDNA microarray analysis mirrored large mounts of genes changed after PAM and PK-15 cells were treated with the individual subunit of HpCDT.In conclusion, we got all 3 subunits of Cdt expressed in soluble form lacking signal peptides, then demonstrate that CdtB has DNase activity in vitro and in vivo; CdtB alone can induce PAM and PK-15 cells G2/M arrest and apoptosis; CdtA or/and CdtC could enhance the ability; tripartite holotoxin exhibits maximum cellular cytotoxicity. Finally, cell apoptosis induced by CDT is p53-dependent. |
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