Evidence of a Substrate-Discriminating Entrance Channel in the Lower Porter Domain of the Multidrug Resistance Efflux Pump AcrB
Evidence of a Substrate-Discriminating Entrance Channel in the Lower Porter Domain of the Multidrug Resistance Efflux Pump AcrB
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DOI：
10.1128/aac.00314-16
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发表时间：
2016-05
影响因子：
4.9
通讯作者：
S. Schuster;M. Vávra;W. Kern
中科院分区：
文献类型：
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作者：
S. Schuster;M. Vávra;W. Kern
摘要耐药细胞分裂转运蛋白家族的外排泵，如大肠杆菌的AcrB，在多药耐药的发展中起着重要作用，但其底物混杂的分子基础尚未完全了解。从一个高度克拉霉素耐药AcrB周质结构域突变体的集合来自体外随机诱变，我们确定了一个异常改变的耐药模式的特点是增加敏感性的许多药物的低分子量，包括氟喹诺酮类，四环素类和恶唑烷酮类，但不变或增加耐药性的药物的高分子量，包括大环内酯类。对14个这样的“分歧抗性”表型突变体和15个对照突变体的测序表明，这种不寻常的表型与残基I38和I671的突变相关，分别主要是苯丙氨酸和苏氨酸，两者都赋予类似的易感性模式。重建的I38F和I671T单突变体，以及工程I38F I671T双突变体与证明外排能力显示了增强或不变的阻力，许多大AcrB基板，但增加了敏感性的几个低分子量的药物，已知结合在远端结合口袋的等效表型。两个异亮氨酸位于邻近的近端结合口袋的底部下方的AcrB的较低的波特域中彼此靠近，可能是由突变损害的优先小药物进入途径的一部分。这一发现支持最近的迹象表明，不同的入口通道所使用的化合物具有不同的物理化学性质，其中分子大小似乎发挥了突出的作用。
ABSTRACT Efflux pumps of the resistance nodulation cell division (RND) transporter family, such as AcrB of Escherichia coli, play an important role in the development of multidrug resistance, but the molecular basis for their substrate promiscuity is not yet completely understood. From a collection of highly clarithromycin-resistant AcrB periplasmic domain mutants derived from in vitro random mutagenesis, we identified variants with an unusually altered drug resistance pattern characterized by increased susceptibility to many drugs of lower molecular weight, including fluoroquinolones, tetracyclines, and oxazolidinones, but unchanged or increased resistance to drugs of higher molecular weight, including macrolides. Sequencing of 14 such “divergent resistance” phenotype mutants and 15 control mutants showed that this unusual phenotype was associated with mutations at residues I38 and I671 predominantly to phenylalanine and threonine, respectively, both conferring a similar susceptibility pattern. Reconstructed I38F and I671T single mutants as well as an engineered I38F I671T double mutant with proved efflux competence revealed an equivalent phenotype with enhanced or unchanged resistance to many large AcrB substrates but increased susceptibility to several lower-molecular-weight drugs known to bind within the distal binding pocket. The two isoleucines located in close vicinity to each other in the lower porter domain of AcrB beneath the bottom of the proximal binding pocket may be part of a preferential small-drug entrance pathway that is compromised by the mutations. This finding supports recent indications of distinct entrance channels used by compounds with different physicochemical properties, of which molecular size appears to play a prominent role.