Plasmid-Mediated Quinolone Resistance

质粒介导的喹诺酮耐药性

• 批准号: 7048494
• 负责人: GEORGE A JACOBY
• 金额: $ 16.55万
• 依托单位: LAHEY CLINIC
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-15 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048494
• 关键词: DNA gyrase; DNA topoisomerases; Escherichia coli; Klebsiella pneumoniae; X ray crystallography; adenosinetriphosphatase; bacterial proteins; chemical binding; crystallization; drug resistance; endopeptidases; enzyme activity; enzyme inhibitors; microorganism culture; molecular cloning; plasmids; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; proteolysis; quinoline; surface plasmon resonance; transfection /expression vector

DESCRIPTION (provided by applicant): 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 in a limited number of isolates in the United States, the qnr gene has recently been discovered in a larger group of isolates from the Far East. The aims of this proposal are: to elucidate the structural requirements for Qnr binding to gyrase, to establish the gyrase activities that Qnr can protect from quinolone inhibition, to determine whether Qnr can also protect the related quinolone target topoisomerase IV, to explore the hypothesis that Qnr blocks quinolone binding to gyrase, to study Qnr binding quantitatively using surface plasmon resonance, to investigate whether Qnr can also protect gyrase from such protein inhibitors as MccB17, CcdB, and Gyrl, to determine whether Qnr and an active gyrase fragment can co-crystallize for structural analysis by x-ray diffraction, and to study the detailed molecular structure of Qnr plasmids to elucidate how this novel property has been acquired. Qnr prevalence will also be explored utilizing samples from parts of the world where quinolone resistance has become common and also from food animal sources. 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.

描述（由申请人提供）：喹诺酮类药物因其抗菌谱广、毒性低以及对其他耐药病原体具有可靠的作用而被广泛使用的抗菌剂。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达到令人震惊的水平。多种染色体突变导致了这种耐药性。长期以来，人们一直认为质粒介导的耐药性不存在。我们发现了一种名为 Qnr 的质粒编码蛋白，它可以保护 DNA 旋转酶免受喹诺酮的抑制。 Qnr 与喹诺酮耐药性的染色体机制相加，属于蛋白质五肽重复家族，通过凝胶置换测定，以不同的亲和力与旋转酶四聚体以及 GyrA 和 GyrB 亚基结合。尽管最初在美国的有限数量的分离株中发现了 qnr 基因，但最近在来自远东的更多分离株中发现了 qnr 基因。该提案的目的是：阐明 Qnr 与促旋酶结合的结构要求，确定 Qnr 可以保护其免受喹诺酮抑制的促旋酶活性，确定 Qnr 是否也可以保护相关的喹诺酮靶点拓扑异构酶 IV，探索以下假设： Qnr 阻断喹诺酮与旋转酶的结合，利用表面等离子共振定量研究 Qnr 结合，研究 Qnr 是否也可以保护旋转酶免受此类蛋白质的影响抑制剂如 MccB17、CcdB 和 Gyrl，以确定 Qnr 和活性旋转酶片段是否可以共结晶，通过 X 射线衍射进行结构分析，并研究 Qnr 质粒的详细分子结构，以阐明如何获得这种新特性。还将利用来自世界上喹诺酮耐药性已普遍存在的地区以及食用动物来源的样本来探讨 Qnr 患病率。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旋转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他蛋白质的活性的细节。