Mechanism and Spread of Qnr-Mediated Resistance

描述(由申请人提供)：QnR介导的耐药性的机制和传播。喹诺酮类药物因其抗菌谱广、毒性低、对耐药病原菌有可靠的抗菌作用而被广泛应用。然而，细菌对喹诺酮类药物的耐药性正在增加，在欧洲和远东的一些地区已经达到令人担忧的水平。各种染色体突变导致了这种耐药性。长期以来，质粒介导的耐药性一直被认为不存在。我们发现了一种名为QnR的质粒编码蛋白，它可以保护DNA旋转酶免受喹诺酮类药物的抑制。QnR与染色体上的喹诺酮耐药机制是相加的，属于五肽重复蛋白家族，通过凝胶置换实验，以不同的亲和力与旋转酶四聚体以及gyrA和gyrB亚基结合。虽然最初在美国的一家医院发现了qnr基因，但最近在来自远东的临床大肠杆菌分离株和来自美国的约10%的耐喹诺酮类肺炎克雷伯菌菌株中发现了qnr基因。本研究的目的是探索QnR阻断喹诺酮类药物与旋转酶结合的假设，通过表面等离子体共振研究QnR与旋转酶的结合动力学，探讨QnR是否也能保护旋转酶不受MccB17、CcdB和Gyr1等蛋白抑制剂的影响，进一步研究QnR的普遍存在及其被质粒获取的遗传基础，并探索QnR是否能与活性旋转酶片段共结晶进行X射线衍射结构分析。这些研究不仅对了解新出现的抗性机制很重要，而且还应该揭示DNA旋转酶和相关的拓扑异构酶如何发挥作用，以及五肽家族中的蛋白质如何与其他蛋白质相互作用并调节其活性。

DESCRIPTION (provided by applicant): Mechanism and Spread of Qnr-Mediated Resistance. Quinolones are widely used antimicrobial agents because of their broad antibacterial spectrum, low toxicity, and reliable action against otherwise resistant pathogens. Bacterial resistance to quinolones, however, is increasing and has reached alarming levels in some parts of Europe and the Far East. Various chromosomal mutations contribute to this resistance. Plasmid-mediated resistance was long thought not to exist. We discovered a plasmid-encoded protein termed Qnr that protects DNA gyrase from quinolone inhibition. Qnr acts additively with chromosomal mechanisms for quinolone resistance, belongs to the pentapeptide repeat family of proteins and, by a gel displacement assay, binds to the gyrase tetramer as well as to the GyrA and GyrB subunits with differing affinities. Although initially found at a single hospital in the United States, the qnr gene has recently been discovered in clinical Escherichia coli isolates from the Far East and in about 10% of quinolone resistant Klebsiella pneumoniae strains from the US. The aims of this proposal are to explore the hypothesis that Qnr blocks quinolone binding to gyrase, to study Qnr binding kinetics by surface plasmon resonance, to investigate whether Qnr can also protect gyrase from such protein inhibitors as MccB17, CcdB, and Gyrl, to study further the prevalence of qnr and the genetic basis of its acquisition by plasmids, and to explore whether Qnr and an active gyrase fragment can co-crystallize for structural analysis by x-ray diffraction. These studies are important not only for understanding an emerging resistance mechanism but should also reveal details of how DNA gyrase and related topoisomerases function and how proteins in the pentapeptide family interact with and regulate the activity of other proteins.