| Cytolethal distending toxin（CDT）is a very important virulence factor for H.parasuis,the pathogenesis of CDT remains largely unknown.To reveal the mechanism of H.parasuis CDT,this research choose NPTr cells as a model to study the function of LRRC8A in H.parasuis CDT induce NPTr cells apoptosis by protein expression and purification,flow cytometry,q RT-PCR,Western Blot and CRISPR/Cas9.The main results of this study are as follows:1. Cdt A,Cdt B and Cdt C subunits successfully expressed,purified and Cdt ABC interact with NPTr cells in vitro.The result showed:Cdt ABC induces dose-dependent apoptosis in NPTr cells（P<0.001）.p-p53,p53,BAX gene expression are upregulated and Bcl-2 gene expression is downregulated in NPTr cells（P<0.01）.Meanwhile,Cdt ABC increases the NPTr cells apoptosis factor caspase 3 activity（P<0.001）.This means H.parasuis Cdt ABC induces NPTr cells apoptosis by p53-dependent.2. To investigate whether Volume regulated anion channel（VRAC）plays a role in H.parasuis Cdt ABC induces apoptosis,VRAC blockers PPQ-102 and NS3728 are used.The result showed:PPQ-102 and NS3728 could salvage NPTr cells from Cdt ABC induces apoptosis（P<0.001）,p-p53,p53 and BAX gene expression are significantly reduced（P<0.001）.In addition,PPQ-102 and NS3728 reduce the apoptosis factor caspase 3 activity in NPTr cells（P<0.001）.These results confirm that VRAC blockers PPQ-102 and NS3728could salvage NPTr cells from Cdt ABC induced apoptosis and initially suggest VRAC may play a key role in H.parasuis Cdt ABC induced apoptosis.3. To explore whether the five members LRRC8A–E of the LRRC8 protein familythat consists VRAC play a role in Cdt ABC interact with NPTr cells,five members LRRC8A–LRRC8E gene expression are dose-dependent upregulated（P<0.01）.This result indicates LRRC8 protein family plays a role in the interaction between H.parasuis Cdt ABC and NPTr cells.4. LRRC8A has been identified as an essential component for VRAC,the NPTr cellswere subjected to si RNA knockdown of LRRC8A expression,p-p53,p53 and BAX gene expression are significantly reduced（P<0.01）.And LRRC8A si RNA reduces the apoptosis factor caspase 3 activity in NPTr cells（P<0.001）.The above results preliminarily indicate that si RNA knockdown of LRRC8A in NPTr cells reduces Cdt ABC induced apoptosis and LRRC8A may play an important role in H.parasuis Cdt ABC induced apoptosis.5. CRISPR/Cas9 technology was used to knockout LRRC8A and obtained LRRC8A–/–cell lines.The result showed:LRRC8A–/–reduces Cdt ABC induces NPTr cells apoptosis（P<0.001）.p-p53,p53 and BAX gene expression are significantly reduced in LRRC8A–/–cells（P<0.001）.Meanwhile,LRRC8A–/–significantly reduces the apoptosis factor caspase 3 activity（P<0.01）.In addition,re-expression of sus scrofa LRRC8A in LRRC8A–/–NPTr cells can effectively restore Cdt ABC induces NPTr cells apoptosis.These data further show,in NPTr cells,the absence of LRRC8A reduces Cdt ABC triggeres apoptosis in NPTr cells and confirm LRRC8A plays a key role in H.parasuis Cdt ABC induced apoptosis.By using VRAC blockers PPQ-102 and NS3728,LRRC8A si RNA to target LRRC8A knockdown and CRISPR/Cas9 technology to target LRRC8A knockout,these three methods all significantly reduce Cdt ABC induces apoptosis.And re-expression of sus scrofa LRRC8A in LRRC8A–/–NPTr cells can effectively restore Cdt ABC induces NPTr cells apoptosis.In conclusion,LRRC8A plays an important role in H.parasuis Cdt ABC induced p53-dependent apoptosis of NPTr cells.It will provide a reference for future studies of LRRC8A in Cdt ABC triggeres cell apoptosis in other Gram-negative bacteria. |
