CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
University of Chinese Academy of Sciences, Beijing, China.
Microbiol Spectr. 2021 Dec 22;9(3):e0074321. doi: 10.1128/Spectrum.00743-21. Epub 2021 Dec 15.
Although glutathione (GSH) has been shown to influence the antimicrobial effects of many kinds of antibiotics, little is known about its role in relation to trimethoprim (TMP), a widely used antifolate. In this study, several genes related to glutathione metabolism were deleted in different Escherichia coli strains (i.e., O157:H7 and ATCC 25922), and their effects on susceptibility to TMP were tested. The results showed that deleting , , , and caused TMP resistance, and deleting also caused resistance to other drugs. Meanwhile, deleting , , and resulted in a significant decrease of the periplasmic glutathione content. Supplementing exogenous GSH or further deleting glutathione importer genes ( and ) restored TMP sensitivity to Δ. Subsequently, the results of quantitative-reverse transcription PCR experiments showed that expression levels of , , and were significantly upregulated in both Δ and Δ. Correspondingly, deleting led to a decreased accumulation of TMP within bacterial cells, and further deleting , , or restored TMP sensitivity to Δ. Inactivation of CpxR and SoxS, two transcriptional factors that modulate the transcription of -, restored TMP sensitivity to Δ. Furthermore, mutations of , , , , and are highly prevalent in E. coli clinical strains. Collectively, these data suggest that reducing the periplasmic glutathione content of E. coli leads to increased expression of - with the involvement of CpxR and SoxS, ultimately causing drug resistance. To the best of our knowledge, this is the first report showing a linkage between periplasmic GSH and drug resistance in bacteria. After being used extensively for decades, trimethoprim still remains one of the key accessible antimicrobials recommended by the World Health Organization. A better understanding of the mechanisms of resistance would be beneficial for the future utilization of this drug. It has been shown that the AcrAB-TolC efflux pump is associated with trimethoprim resistance in E. coli clinical strains. In this study, we show that E. coli can sense the periplasmic glutathione content with the involvement of the CpxAR two-component system. As a result, reducing the periplasmic glutathione content leads to increased expression of , , and via CpxR and SoxS, causing resistance to antimicrobials, including trimethoprim. Meanwhile, mutations in the genes responsible for periplasmic glutathione content maintenance are highly prevalent in E. coli clinical isolates, indicating a potential correlation of the periplasmic glutathione content and clinical antimicrobial resistance, which merits further investigation.
虽然谷胱甘肽 (GSH) 已被证明会影响许多种类抗生素的抗菌作用，但关于其与广泛使用的抗叶酸药物甲氧苄啶 (TMP) 之间的关系知之甚少。在这项研究中，我们在不同的大肠杆菌菌株（即 O157:H7 和 ATCC 25922）中删除了几个与谷胱甘肽代谢相关的基因，并测试了它们对 TMP 敏感性的影响。结果表明，删除 、 、 和 导致 TMP 耐药，删除 也导致对其他药物的耐药性。同时，删除 、 和 导致周质谷胱甘肽含量显著降低。添加外源性 GSH 或进一步删除谷胱甘肽导入基因（ 和 ）可使 Δ 恢复对 TMP 的敏感性。随后，定量逆转录 PCR 实验结果表明，Δ 和 Δ 中 、 和 的表达水平均显著上调。相应地，删除 导致细菌细胞内 TMP 的积累减少，进一步删除 、 、 或 可使 Δ 恢复对 TMP 的敏感性。CpxR 和 SoxS 这两个调节 - 转录的转录因子的失活可使 Δ 恢复对 TMP 的敏感性。此外，在大肠杆菌临床株中， 、 、 、 和 的突变非常普遍。总的来说，这些数据表明，降低大肠杆菌周质谷胱甘肽含量会导致 CpxR 和 SoxS 参与的 - 表达增加，最终导致耐药性。据我们所知，这是首次报道周质 GSH 与细菌耐药性之间存在关联。甲氧苄啶在被广泛使用了几十年后，仍然是世界卫生组织推荐的关键可获得的抗菌药物之一。更好地了解耐药机制将有助于该药物的未来应用。已经表明，AcrAB-TolC 外排泵与大肠杆菌临床株中的 TMP 耐药性有关。在这项研究中，我们表明，大肠杆菌可以通过 CpxAR 双组分系统感知周质谷胱甘肽含量。因此，降低周质谷胱甘肽含量会导致 CpxR 和 SoxS 介导的 - 、 和 的表达增加，导致对抗生素（包括 TMP）的耐药性。同时，负责维持周质谷胱甘肽含量的基因发生突变在大肠杆菌临床分离株中非常普遍，表明周质谷胱甘肽含量与临床抗菌耐药性之间存在潜在相关性，值得进一步研究。
