Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae
Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae
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DOI：
10.1074/jbc.ra117.000383
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发表时间：
2018-04-13
影响因子：
4.8
通讯作者：
Yoon, Sang Sun
中科院分区：
文献类型：
--
作者：
Kim, Hwa Young;Go, Junhyeok;Yoon, Sang Sun
霍乱弧菌是霍乱的病原体。耐药性霍乱弧菌菌株的出现正在增加，但其潜在机制仍不清楚。在此，我们报告了严格的反应调节剂和应激警报素鸟苷四磷酸和五磷酸（（p）ppGpp）显着有助于霍乱弧菌的抗生素耐受性。我们发现，N16961，一种流行性霍乱弧菌菌株，及其等基因（p）ppGpp-过表达突变体Delta relA Delta spoT都比（p）ppGpp（o）（Delta relA Delta relV Delta spoT）和Delta dksA突变体更耐抗生素，后者分别不能产生或利用（p）ppGpp。我们还发现，（p）ppGpp（o）突变体中编码乌头酸酶B和三羧酸（TCA）循环基因acnB的额外破坏增加了其抗生素耐受性。此外，TCA循环基因（包括acnB）的表达在（p）ppGpp（o）中增加，但在抗生素抗性Delta relA Delta spoT突变体中没有增加，表明（p）ppGpp抑制TCA循环活性，从而导致抗生素抗性。重要的是，当在厌氧条件下生长或与铁螯合剂一起孵育时，（p）ppGpp（o）突变体变得耐缺氧，这表明活性氧（ROS）参与了缺氧介导的细菌杀伤。与这一假设相一致，四环素处理显著增加了敏感突变体中ROS的产生。有趣的是，Fe（III）ABC转运蛋白底物结合蛋白FbpA的表达在（p）ppGpp（o）中增加了10倍，并且fbpA基因缺失恢复了四环素暴露的（p）ppGpp（o）细胞的活力。值得注意的是，在（p）ppGpp积累突变体中，FbpA表达被抑制，导致细胞内游离铁的减少，这是产生ROS的芬顿反应所需的。我们的研究结果表明，（p）ppGpp介导的中枢代谢和铁摄取的抑制减少了霍乱弧菌中的维生素C诱导的氧化应激。
The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosinetetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae. We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant Delta relA Delta spoT are both more antibiotic-resistant than (p)ppGpp(o) (Delta relA Delta relV Delta spoT) and Delta dksA mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene acnB in the (p)ppGpp(o) mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including acnB, was increased in (p)ppGpp(o), but not in the antibiotic-resistant Delta relA Delta spoT mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp(o) mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp(o), and fbpA gene deletion restored viability of tetracycline-exposed (p)ppGpp(o) cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in V. cholerae.