From the Department of Microbiology and Immunology, Brain Korea 21 PLUS Project for Medical Science, and.
the Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine, Seoul 03722, Korea and.
J Biol Chem. 2018 Apr 13;293(15):5679-5694. doi: 10.1074/jbc.RA117.000383. Epub 2018 Feb 23.
The pathogen is the causative agent of cholera. Emergence of antibiotic-resistant strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in We found that N16961, a pandemic strain, and its isogenic (p)ppGpp-overexpressing mutant ΔΔ are both more antibiotic-resistant than (p)ppGpp (ΔΔΔ) and Δ 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 in the (p)ppGpp mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including , was increased in (p)ppGpp, but not in the antibiotic-resistant ΔΔ 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 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, and gene deletion restored viability of tetracycline-exposed (p)ppGpp 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 .
病原体是霍乱的致病因子。抗生素耐药菌株的出现正在增加，但潜在机制尚不清楚。在此，我们报告称，严格反应调节剂和应激警报素鸟苷四磷酸和五磷酸（（p）ppGpp）显著有助于 中抗生素的耐受性。我们发现，N16961，一种大流行株，及其同源（p）ppGpp 过表达突变体 ΔΔ比（p）ppGpp（ΔΔΔ）和 Δ 突变体更能抵抗抗生素，分别不能产生或利用（p）ppGpp。我们还发现，在（p）ppGpp 突变体中额外破坏 aconitase B 编码和三羧酸（TCA）循环基因 会增加其抗生素耐受性。此外，TCA 循环基因的表达，包括 ，在（p）ppGpp 中增加，但在抗生素抗性 ΔΔ 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，当在厌氧条件下或与铁螯合剂孵育时，（p）ppGpp 突变体变得对抗生素具有耐受性，表明活性氧物种（ROS）参与了抗生素介导的细菌杀伤。与该假说一致，四环素处理显著增加了抗生素敏感突变体中的 ROS 产生。有趣的是，（p）ppGpp 中表达的 Fe（III）ABC 转运体底物结合蛋白 FbpA 增加了 10 倍，并且 基因缺失恢复了暴露于四环素的（p）ppGpp 细胞的活力。值得注意的是，在（p）ppGpp 积累突变体中，FbpA 的表达受到抑制，导致用于产生 ROS 的 Fenton 反应的细胞内游离铁减少。我们的结果表明，（p）ppGpp 介导的中心代谢和铁摄取的抑制降低了 中的抗生素诱导的氧化应激。
