多黏菌素A1介导耐药铜绿假单胞菌羟自由基致死的机理

Mechanism of polymyxin A1-mediated hydroxyl radical lethality in drug-resistant Pseudomonas aeruginosa

摘要:

【背景】多黏菌素被誉为对抗多重耐药菌感染的最后一道防线，而多黏菌素A1是该家族中很少被探究的同系物，其对耐药性铜绿假单胞菌(Pseudomonas aeruginosa) PAO1具有良好的杀菌效果。【目的】探究对P. aeruginosa PAO1的杀菌机理。【方法】首先通过荧光探针测定了多黏菌素A1处理后细胞内羟自由基变化；然后通过添加硫脲和联吡啶探究对多黏菌素A1介导的杀菌效果和羟自由基产生的影响；接着通过表型分析细胞内氧自由基(reactive oxygen species, ROS)、脂质过氧化物、DNA损伤、总铁离子和亚铁离子变化来表征羟自由基引起的死亡；最后利用实时荧光定量PCR (real time fluorescence quantitative PCR, RT-qPCR)分析通路基因表达来进一步验证羟自由基致死机理。【结果】多黏菌素A1处理引起P. aeruginosa PAO1羟自由基产生，硫脲和联吡啶可以有效缓解多黏菌素A1对细胞的致死效果并降低羟自由基含量；多黏菌素A1处理后细胞内ROS和脂质氧化水平提高，同时引起DNA损伤和亚铁离子的紊乱；此外，细胞ROS清除酶基因、铁摄取和铁硫簇修复相关基因，以及DNA损伤修复基因表达水平均显著提高。【结论】多黏菌素A1对耐药性铜绿假单胞菌的一种致死机理可能是介导细胞通过芬顿反应产生羟自由基，羟自由基损伤DNA、脂质从而导致细胞死亡。

Abstract:

[Background] Polymyxins are considered as the last line of defense against multidrug-resistant (MDR) infections, and polymyxin A1 with a bactericidal effect against MDR Pseudomonas aeruginosa PAO1 is a rarely studied homolog in this family. [Objective] To explore the effect of polymyxin A1 on P. aeruginosa PAO1. [Methods] A fluorescent probe was used to measure the hydroxyl radical level in the cells treated with polymyxin A1. Then, thiourea and bipyridine were added, and their effects on the bactericidal effect of polymyxin A1 and the production of intracellular hydroxyl radicals were investigated. Subsequently, hydroxyl radical-induced death was characterized in terms of reactive oxygen species (ROS), lipid peroxides, DNA damage, and changes in total iron and ferrous iron ions. Finally, RT-qPCR was employed to determine the expression of genes involved in hydroxyl radical lethality. [Results] The level of hydroxyl radicals in P. aeruginosa PAO1 was increased by the treatment with polymyxin A1, and the addition of thiourea and bipyridine lowered the level of hydroxyl radicals and attenuated the lethality. Polymyxin A1 elevated the levels of lipid oxidation and ROS, damaged bacterial DNA, and caused a disorder of ferrous iron ion. In addition, the expression levels of genes encoding ROS scavenging enzymes, iron uptake and iron-sulfur cluster repair, and DNA repair were up-regulated in the cells treated with polymyxin A1. [Conclusion] Polymyxin A1 may kill MDR P. aeruginosa PAO1 by regulating the production of hydroxyl radicals through the Fenton reaction, which damaged DNA and lipids and led to cell death.