Allele-based analysis revealed the critical functions of region 277–297 in the NorA efflux pump of Staphylococcus aureus
Allele-based analysis revealed the critical functions of region 277–297 in the NorA efflux pump of Staphylococcus aureus
复制标题
基于等位基因的分析揭示了金黄色葡萄球菌 NorA 外排泵中 277–297 区域的关键功能
DOI：
10.1093/jac/dkab066
复制
发表时间：
2021
影响因子：
5.2
通讯作者：
Chunyu Yang
中科院分区：
文献类型：
--
作者：
Yan Shang;Peiwen Lv;Shannan Li;Wenkai Wang;Yuanxiang Liu;Chunyu Yang
抽象的。目标。金黄色葡萄球菌中的 NorA 外排泵介导对许多氟喹诺酮 (FQ) 抗生素的耐药性。在表现出不同 FQ 抗性特征的各种金黄色葡萄球菌菌株中发现了三个具有高度序列相似性的norA等位基因。本研究旨在阐明这三种等位基因变异的不同外排活性的潜在分子基础。分析了 20 个金黄色葡萄球菌分离株的norA 基因型。进行多重比对和保守分析来探索进化变异。在大肠杆菌中异源表达后，构建了七个突变体用于 MIC 测试、外排活性和构象变化测量。结果。鉴定出三个 NorA 等位基因，它们表现出不同的 FQ MIC 和不同的溴化乙锭外排活性，其中 NorAII 蛋白显示出最强的活性。通过保守分析，在3个等位肽中共鉴定出29个单氨基酸多态性，其中7个位点密集分布在277-297区域。 NorAII中这7个残基的突变均显着损害了耐药性和外排活性，并且三个关键突变体在荧光共振能量转移（FRET）分析中显示出构象变化。结论。 277-297区域的进化变异可能是三个norA等位基因功能差异的主要解释，并可作为开发新型NorA抑制剂的潜在靶点。
Abstract. Objectives. The NorA efflux pump in Staphylococcus aureus mediates resistance to many fluoroquinolone (FQ) antibiotics. Three norA alleles with high sequence similarity are found in various S. aureus strains exhibiting different FQ resistance profiles. This study aimed to elucidate the underlying molecular basis for the varying efflux activity of these three allelic variations.. Methods. The norA genotypes of 20 S. aureus isolates were analysed. Multiple alignments and conservative analyses were conducted to explore the evolutionary variations. After heterologous expression in Escherichia coli, seven mutants were constructed for MIC tests, efflux activity and conformational change measurements.. Results. Three NorA alleles were identified that displayed different FQ MICs and varying efflux activity for ethidium bromide, with the NorAII protein showing the strongest activity. A total of 29 single amino acid polymorphisms were identified by conservative analysis within three allelic peptides, with seven sites densely distributed in the 277–297 region. Mutations of these seven residues in NorAII all significantly impaired drug resistance and efflux activity, and three key mutants showed conformational changes in fluorescence resonance energy transfer (FRET) analysis.. Conclusions. Evolutionary variations of the 277–297 region could be a major explanation for the functional difference of three norA alleles and serve as a potential target for the development of novel NorA inhibitors.