The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication
The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication
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DOI：
10.1128/mbio.01127-19
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发表时间：
2019-05-01
期刊：
影响因子：
6.4
通讯作者：
Isberg, Ralph R.
中科院分区：
文献类型：
--
作者：
Geisinger, Edward;Vargas-Cuebas, German;Isberg, Ralph R.
氟喹诺酮类抗生素耐药性的出现限制了该类抗生素的临床疗效。在鲍曼不动杆菌中，由于gyrA（DNA旋转酶）和parC（拓扑异构酶IV [topo IV]）的突变，大多数临床分离株目前显示出高水平的耐药性。为了研究氟喹诺酮耐药的分子基础，在药物敏感和耐药菌株中进行了详尽的突变分析，以确定改变环丙沙星敏感性的位点。为此，在编码药物靶标的基因中具有各种等位基因的菌株中进行了超过60，000个独特插入突变的平行适应性测试。发现改变药物敏感性的突变谱在药物敏感性和gyrA parC双突变背景中是相似的，在两个基因中都具有抗性等位基因。相比之下，引入一个单一的gyrA抗性等位基因，导致环丙沙星优先中毒的拓扑异构酶IV，导致极端的改变插入突变的健身景观。优先拓扑异构酶IV中毒的显着特点是增强诱导的DNA合成的区域中的两个内源性原噬菌体，与DNA合成相关的切除和环化的DNA。在gyrA背景中的选择性DNA合成的诱导也与相对于在野生型（WT）或gyrA parC双突变体中观察到的增强的原噬菌体基因转录和增强的致突变SOS响应的激活有关。因此，导致高环丙沙星耐药性逐步进化的突变的积累与SOS反应和内源性原噬菌体DNA合成的调节密切相关。重要性氟喹诺酮类药物由于能够靶向对DNA复制至关重要的多种细菌酶，即拓扑异构酶DNA促旋酶和拓扑异构酶IV，因此已成为非常成功的抗生素。不幸的是，降低药物对这两种酶的亲和力的突变现在很普遍，使得这些药物对许多病原体无效。为了破坏这种形式的耐药性，我们研究了具有靶标改变的细菌如何不同地科普氟喹诺酮暴露。我们在医院内病原体A中研究了这个问题。鲍曼不动杆菌，导致抗药性的危及生命的感染。采用全基因组方法，我们发现了许多途径，可以利用这些途径来提高氟喹诺酮敏感性，而不依赖于靶标的改变。值得注意的是，氟喹诺酮靶向特定突变体中的topo IV引起了原噬菌体复制的显著过度诱导，并增强了诱变DNA损伤反应，但这些反应在以DNA促旋酶为主要靶标的菌株中被抑制。这项工作表明，通过靶向修饰的耐药性进化可以深刻地调节抗生素应激反应，揭示潜在的耐药性相关责任。
The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis.IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the to-poisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes drugresistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.