spo0A对艰难梭菌毒素A/B蛋白表达的调节作用

The regulatory role of spo0A in Clostridioides difficile toxin A and toxin B production

目的研究spo0A基因对艰难梭菌毒素A和毒素B蛋白表达的调控作用。方法采用ClosTron基因敲除技术构建艰难梭菌C25菌株spo0A基因突变模型。实时定量反转录PCR方法测不同生长时期(培养后5、12、24、48 h)突变株和亲代株的毒素基因tcdA、tcdB和毒素调控基因tcdC、tcdR、tcdE的表达量,分别用酶联免疫吸附试验(ELISA)和细胞毒中和试验测定细菌培养液中毒素A和毒素B的含量。结果C25菌株spo0A基因突变株在培养5、12、24 h后,其毒素A和毒素B的含量均明显高于亲代株,培养后48 h突变株毒素A含量高于亲代株,毒素B含量与亲代株无明显差异。spo0A基因突变株在培养12、24、48 h后,tcdA、tcdB、tcdE、tcdR基因表达量均高于亲代株;突变菌株tcdC基因的表达无明显高于或低于亲代株。结论spo0A基因对艰难梭菌毒素A和毒素B的表达起负调控作用,其作用可能与抑制tcdE、tcdR基因的表达相关。

Objective To investigate the regulatory effect of spo0A gene in the production of Clostridioides difficile toxin A and toxin B.Methods ClosTron system was used to construct the spo0A gene mutation of C.difficile C25.The expression levels of toxin genes tcdA,tcdB and toxin regulatory genes tcdC,tcdR and tcdE were measured by real-time PCR at different growth stages(5 h,12 h,24 h and 48 h after culture).The concentrations of toxin A and toxin B in culture supernatants were determined by ELISA and Vero cell cytotoxicity assay,respectively.Results The spo0A mutant strain produced significantly higher concentration of toxin A and toxin B than the parental strain after 5 h,12 h and 24 h growth.The mutant strain produced higher concentration of toxin A but not toxin B than the parental strain after 48 h growth.The expression levels of tcdA,tcdB,tcdE and tcdR gene were higher in the spo0A mutation stain than in the parental strain after 12 h,24 h and 48 h growth,but no difference was found for the expression level of gene tcdC.Conclusions Spo0A gene negatively regulates the expression of Clostridioides difficile toxin A and toxin B.The effect might be related to the inhibition of the expression of tcdE and tcdR.