Rajapaksha Prasangi, Ojo Isoiza, Yang Ling, Pandeya Ankit, Abeywansha Thilini, Wei Yinan
Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
Antibiotics (Basel). 2021 Jul 8;10(7):830. doi: 10.3390/antibiotics10070830.
The RND family efflux pump AcrAB-TolC in and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called "dominant negative" effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.
大肠杆菌中的RND家族外排泵AcrAB - TolC及其在其他革兰氏阴性菌中的同源物是赋予细胞多重耐药性的主要因素。尽管通过晶体学和冷冻电镜技术，泵复合物的结构解析分辨率不断提高，但动态组装过程仍知之甚少。在此，我们通过在野生型大肠杆菌细胞中过表达功能缺陷的泵组件来测试其对泵组装过程的影响。引入缺陷组件预计会降低复合物的外排效率，从而产生所谓的“显性负效应”。作为研究最深入的细菌多重耐药外排泵之一，已有许多关于AcrA和AcrB突变的报道，这些突变通过不同机制破坏外排。我们研究了五组在组装和底物外排不同方面存在缺陷的AcrB和AcrA突变体。我们发现，即使它们的表达浓度比基因组对应物高出许多倍，也没有一个表现出预期的显性负效应。AcrAB - TolC复合物的组装似乎有一种校对机制，能有效消除无效泵复合物的形成。
