Understanding DNA transport by topo IV and gyrase to counter antimicrobial resistance

抗生素耐药性是一个世界性的问题，也是感染治疗的主要挑战。细菌正在对我们最重要的抗生素产生抗药性，甚至危及简单的医疗程序。一个严重的威胁是肺炎链球菌，一种全球性的病原体，导致危及生命的肺炎和脑膜炎的儿童和老年人。越来越多，S。发现肺炎分离株对青霉素和氟喹诺酮类抗生素具有耐药性，而这两种抗生素是我们抗生素库中的关键成员。众所周知，氟喹诺酮类药物通过促旋酶和拓扑异构酶（topo）IV干扰DNA断裂，这两种酶解开DNA，是细菌DNA复制和生长所需的。值得注意的是，这些酶在DNA中产生短暂的断裂，并通过断裂与另一个DNA交叉。通过这种方式，当DNA在细胞中复制时，旋转酶可以消除扭曲，而拓扑异构酶IV可以在细胞分裂之前解开缠结的染色体。通过使用X射线晶体学来解决这些与DNA和氟喹诺酮类结合的酶的结构，我们现在详细了解了喹诺酮类如何结合和捕获DNA断裂以及耐药性如何产生。然而，几乎没有人知道被运输的DNA的性质，它是如何被识别，捕获，然后穿过DNA断裂的。为了填补这一知识空白，并在我们最近的拓扑IV转运DNA复合物结构的指导下，我们将使用X射线晶体学，冷冻电子显微镜，荧光和蛋白质生物化学等技术的组合来建立由拓扑IV和螺旋酶从S.肺炎克雷伯氏菌（Klebsiella pneumoniae）和肺炎克雷伯氏菌（Klebsiella pneumoniae），另一种对喹诺酮高度耐药的病原体。这项工作的完成将对理解这些迷人的分子机器做出重大贡献，并将为设计通过靶向DNA转运克服耐药性的新药提供新的机会。新药对于解决全球抗生素耐药性紧急情况至关重要。

Antibiotic resistance is a worldwide problem and a major challenge for the treatment of infection. Bacteria are becoming resistant to our most important antibiotics jeopardising even straightforward medical procedures. One serious threat is posed by Streptococcus pneumoniae, a global pathogen that causes life-threatening pneumonia and meningitis in childern and the elderly. Increasingly, S. pneumoniae isolates are found to be resistant to penicillins and to fluoroquinolones, which are key members of our antibiotic arsenal. It is known that fluoroquinolones interfere with DNA breakage by gyrase and topoisomerase (topo) IV, two enzymes that untangle DNA and are required for bacterial DNA replication and growth. Remarkably, these enzymes make a transient break in DNA and cross another DNA through the break. By this means, gyrase can remove twists when DNA is copied in the cell, and topo IV can unlink tangled chromosomes ahead of cell division. By using X-ray crystallography to solve the structure of these enzymes bound to DNA and fluoroquinolones, we now understand in detail how quinolones bind and trap the DNA break and how resistance arises. However, almost nothing is known about the nature of the transported DNA, how it is recognised, captured and then crossed through the DNA break. To fill this gap in knowledge, and guided by our recent structure of a topo IV-transport DNA complex, we shall use a combination of techniques including X-ray crystallography, cryo-electron microscopy, fluorescence and protein biochemistry to establish the mechanism of DNA transport by topo IV and gyrase from S. pneumoniae and from Klebsiella pneumoniae, another pathogen highly resistant to quinolones. Completion of the work will be a major contribution in understanding these fascinating molecular machines and will provide new opportunites for design of new drugs that overcome resistance by targeting DNA transport. New drugs will be essential in addressing the global emergency of antimicrobial resistance.