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。喹诺酮类药物是广泛使用的抗菌药物，因为它们具有广谱抗菌谱、低毒性和对其他耐药病原体的可靠作用。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了令人震惊的水平。各种染色体突变导致了这种耐药性。质粒介导的耐药性长期以来被认为是不存在的。我们发现了一个质粒编码的蛋白质称为Qnr，保护DNA促旋酶从喹诺酮抑制。Qnr与喹诺酮耐药的染色体机制相加作用，属于五肽重复蛋白家族，并且通过凝胶置换测定，与促旋酶四聚体以及具有不同亲和力的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，但最近在远东的临床大肠杆菌分离株和美国约10%的喹诺酮耐药肺炎克雷伯菌菌株中发现了qnr基因。本提案的目的是探索Qnr阻断喹诺酮与促旋酶结合的假设，通过表面等离子体共振研究Qnr结合动力学，研究Qnr是否也可以保护促旋酶免受MccB 17、CcdB和Gyrl等蛋白质抑制剂的影响，进一步研究qnr的流行及其通过质粒获得的遗传基础，并探索Qnr和活性促旋酶片段是否可以共结晶以通过X射线衍射进行结构分析。这些研究不仅对于理解新出现的耐药机制很重要，而且还应该揭示DNA促旋酶和相关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的细节。

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.