Critical Role of 3'-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3).
Critical Role of 3'-Downstream Region of pmrB in Polymyxin Resistance in Escherichia coli BL21(DE3).
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DOI：
10.3390/microorganisms9030655
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发表时间：
2021-03-22
期刊：
影响因子：
4.5
通讯作者：
Lin J
中科院分区：
文献类型：
--
作者：
Xu F;Hinenoya A;Zeng X;Li XP;Guan Z;Lin J
多粘菌素，如粘菌素和多粘菌素B，是作为治疗人类多重耐药革兰氏阴性细菌感染的最后手段使用的药物。粘菌素耐药性的增加已对人类健康构成严重威胁，阻碍了其机制的深入研究。在这项研究中，使用功能性克隆的方法，我们研究了大肠杆菌BL 21（DE 3）的黏菌素耐药性的分子基础。5个插入片段大小在3.8 ~ 10.7kb之间的转化子表现出明显的粘菌素抗性增强，其中3个含有pmrB位点，2个含有pmrD位点。逐步亚克隆结果表明，具有单一G361 A突变的pmrB和至少103 bp的pmrB下游区域是赋予粘菌素抗性所必需的。mRNA水平和稳定性的分析表明，下游区域的长度极大地影响pmrB mRNA水平，但不影响其半衰期。通过质谱法进行的脂质A分析显示，含有具有较长下游区域（103或126 bp）的pmrB的构建体在脂质A中具有电荷改变的1 -4-氨基阿拉伯糖（Ara 4 N）和磷酸乙醇胺（pEtN）修饰，这在载体对照和含有具有86 bp下游区域的pmrB的构建体中均未观察到。总之，本研究的结果表明，pmrB的3′-下游区域是PmrB介导的脂质A修饰和大肠杆菌粘菌素耐药性的关键。coli BL 21（DE 3）中，PmrB介导的黏菌素抗性可能是一种新的调控机制。杆菌
Polymyxins, such as colistin and polymyxin B, are the drugs used as a last resort to treat multidrug-resistant Gram-negative bacterial infections in humans. Increasing colistin resistance has posed a serious threat to human health, warranting in-depth mechanistic research. In this study, using a functional cloning approach, we examined the molecular basis of colistin resistance in Escherichia coli BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing pmrB locus and two containing pmrD locus. Stepwise subcloning indicated that both the pmrB with a single G361A mutation and at least a 103 bp downstream region of pmrB are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the pmrB mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing pmrB with a longer downstream region (103 or 126 bp) have charge-altering l-4-aminoarabinose (Ara4N) and phosphoethanolamine (pEtN) modifications in lipid A, which were not observed in both vector control and the construct containing pmrB with an 86 bp downstream region. Together, the findings from this study indicate that the 3′-downstream region of pmrB is critical for the PmrB-mediated lipid A modifications and colistin resistance in E. coli BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in E. coli.
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影响因子：
3.6
作者：
Gunn, JS;Lim, KB;Miller, SI
通讯作者：
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影响因子：
11.4
作者：
Kox, LFF;Wösten, MMSM;Groisman, EA
通讯作者：
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影响因子：
4.9
作者：
Lin, J;Michel, LO;Zhang, QJ
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Zhang, QJ
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作者：
Kato, Akinori;Chen, H. Deborah;Latifi, Tammy;Groisman, Eduardo A.
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Groisman, Eduardo A.
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL