TpbA通过调节c-di-GMP抑制鲍曼不动杆菌ATCC17978生物膜的形成

TpbA inhibits biofilm formation of acinetobacter baumannii ATCC17978 by regulating c-di-GMP

目的分析生物膜形成相关的酪氨酸磷酸酶A(TpbA)经调节3,5-环鸟苷二磷酸(c-di-GMP)影响鲍曼不动杆菌ATCC17978生物膜的形成。方法通过上海生工合成TpbA基因序列,构建pET-28a-TpbA载体,原核重组表达目的蛋白TpbA经亲和层析纯化后并用western blot验证,将该载体转入鲍曼不动杆菌ATCC17978,采用western blot和磷酸酶活性实验鉴定TpbA表达及活性,分析c-di-GMP表达水平及生物膜形成情况。结果在成功构建pET-28a-TpbA载体并证实表达TpbA的基础上,将该载体转入鲍曼不动杆菌ATCC17978,western blot表明TpbA过表达(P<0.05),磷酸酶活性实验证实过表达的TpbA具有酶活性(P<0.05)。进一步分析发现,ATCC17978中TpbA过表达能够降低c-di-GMP的水平(P<0.05),表明TpbA能够有效调节鲍曼不动杆菌的c-di-GMP。ATCC17978中c-di-GMP相关的生物膜形成在TpbA过表达作用下能够被显著抑制(P<0.05)。结论本研究初步证实了TpbA通过调节c-di-GMP,可以抑制ATCC17978生物膜形成,为抑制鲍曼不动杆菌生物膜的形成提供了新的潜在靶点和实验数据。

Objective To analyze the effects of TpbA(Tyrosine phosphatase related to biofilm formation A)on biofilm of acinetobacter baumannii via regulating 3,5-cyclic diguanylate(c-di-GMP).Methods The TpbA gene sequence was synthesized by Shanghai Sangong,and then the pET-28a-TpbA vector was constructed and the target protein TpbA was expressed prokaryotically.After purification by affinity chromatography and western blot verification,the vector was transformed into Acinetobacter baumannii ATCC17978,and the expression and activity of TpbA were identified by western blot and phosphatase activity assay,and the expression level of c-di-GMP and biofilm formation were analyzed.Results On the basis of successfully constructed the pET-28a-TpbA vector and confirmed the expression of TpbA,the vector was transformed into Acinetobacter baumannii ATCC17978,western blot showed that TpbA was overexpressed(P<0.05),and the phosphatase activity experiment confirmed the overexpressed TpbA Has enzymatic activity(P<0.05).Further analysis found that the overexpression of TpbA in ATCC17978 reduced the level of c-di-GMP(P<0.05),indicating that TpbA can effectively regulate the c-di-GMP of Acinetobacter baumannii.The c-di-GMP-related biofilm formation in ATCC17978 was significantly inhibited by overexpression of TpbA(P<0.05).Conclusion This study preliminarily confirms that TpbA inhibited the biofilm formation of ATCC17978 by regulating c-di-GMP,which could provide new potential targets and experimental data for inhibiting the biofilm formation of acinetobacter baumannii.