Rajapaksha Prasangi, Pandeya Ankit, Wei Yinan
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, United States.
ACS Omega. 2020 Aug 20;5(34):21844-21852. doi: 10.1021/acsomega.0c02921. eCollection 2020 Sep 1.
The resistant-nodulation-division (RND) superfamily member tripartite AcrA-AcrB-TolC efflux pump is a major contributor to the multidrug resistance in AcrB is the inner membrane protein of the efflux complex and is responsible for the recognition and binding of compounds before their transportation out of the cell. Understanding the dynamics of AcrB during functional rotation in the process of drug efflux is the focus of this study. For this purpose, we introduced six inter-subunit disulfide bonds into the periplasmic domain of AcrB using site-directed mutagenesis to study the importance of the relative flexibility at the inter-subunit interface. Western blot analysis revealed the formation of disulfide bond-linked AcrB oligomers, which were reduced into monomers under reducing conditions. The impact of mutation and formation of disulfide bond on efflux were evaluated via comparison of the minimum inhibitory concentration (MIC) of an knockout strain expressing different mutants. The double Cys mutants tested led to equal or higher susceptibility to AcrB substrates compared to their corresponding single mutants. To determine if the reduction of activity in a double mutant is due to restriction on conformational changes by the disulfide bond formation, ethidium bromide accumulation assays were conducted utilizing dithiothreitol (DTT) as the reducing agent. In two cases, the activities of the double Cys mutants were partially restored by DTT reduction, confirming the importance of relative movement in the respective location for function. These findings provide new insights into the dynamics of the AcrAB-TolC efflux pump in .
耐药-结瘤-分裂（RND）超家族成员三联体AcrA-AcrB-TolC外排泵是大肠杆菌多重耐药的主要原因。AcrB是外排复合物的内膜蛋白，负责化合物在转运出细胞之前的识别和结合。了解AcrB在药物外排过程中功能旋转期间的动力学是本研究的重点。为此，我们通过定点诱变在AcrB的周质结构域中引入了六个亚基间二硫键，以研究亚基间界面处相对灵活性的重要性。蛋白质免疫印迹分析显示形成了二硫键连接的AcrB寡聚体，其在还原条件下被还原为单体。通过比较表达不同突变体的大肠杆菌敲除菌株的最低抑菌浓度（MIC），评估突变和二硫键形成对外排的影响。与相应的单突变体相比，所测试的双半胱氨酸突变体导致对AcrB底物的敏感性相同或更高。为了确定双突变体中活性的降低是否是由于二硫键形成对构象变化的限制，使用二硫苏糖醇（DTT）作为还原剂进行溴化乙锭积累试验。在两种情况下，双半胱氨酸突变体的活性通过DTT还原而部分恢复，证实了各自位置上相对运动对功能的重要性。这些发现为大肠杆菌中AcrAB-TolC外排泵的动力学提供了新的见解。
