Xu Fuzhou, Hinenoya Atsushi, Zeng Ximin, Li Xing-Ping, Guan Ziqiang, Lin Jun
Department of Animal Science, University of Tennessee, Knoxville, TN 37996, USA.
Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China.
Microorganisms. 2021 Mar 22;9(3):655. doi: 10.3390/microorganisms9030655.
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 BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing locus and two containing locus. Stepwise subcloning indicated that both the with a single G361A mutation and at least a 103 bp downstream region of are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing 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 with an 86 bp downstream region. Together, the findings from this study indicate that the 3'-downstream region of is critical for the PmrB-mediated lipid A modifications and colistin resistance in BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in .
多粘菌素，如黏菌素和多粘菌素B，是用于治疗人类多重耐药革兰氏阴性菌感染的最后手段。黏菌素耐药性的增加对人类健康构成了严重威胁，需要进行深入的机制研究。在本研究中，我们使用功能克隆方法研究了BL21(DE3)中黏菌素耐药性的分子基础。五个插入片段大小在3.8至10.7 kb之间的转化子显示出显著增加的黏菌素耐药性，其中三个含有 基因座，两个含有 基因座。逐步亚克隆表明，具有单个G361A突变的 和 至少103 bp的下游区域对于赋予黏菌素耐药性都是必不可少的。对mRNA水平和稳定性的分析表明，下游区域的长度极大地影响了 mRNA水平，但不影响其半衰期。通过质谱进行的脂质A分析表明，含有较长下游区域（103或126 bp）的 构建体在脂质A中具有电荷改变的l-4-氨基阿拉伯糖（Ara4N）和磷酸乙醇胺（pEtN）修饰，而在载体对照和含有86 bp下游区域的 构建体中均未观察到这种修饰。总之，本研究结果表明， 的3'下游区域对于BL21(DE3)中PmrB介导的脂质A修饰和黏菌素耐药性至关重要，提示了 中PmrB介导的黏菌素耐药性的一种新调控机制。
