Plasmid-mediated Quinolone resistance

质粒介导的喹诺酮类耐药

基本信息

批准号：
8475422
负责人：
David C Hooper
金额：
$ 38.5万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-01-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8475422
关键词：
Address Alanine Algae Alleles Amino Acids Antibiotic Resistance Bacteria Bacterial Chromosomes Binding Biological Models Calorimetry Categories Cells Chromosomes Ciprofloxacin Complex Crystallography DNA DNA Binding DNA Damage DNA Gyrase DNA Topoisomerase IV Dissection Elements Enzyme Inhibition Enzymes Family Genes Gram-Negative Bacteria Growth Homologous Gene Human Hybrids In Vitro Integrons Link Measures Mediating Medical Mobile Genetic Elements Multi-Drug Resistance Multidrug Resistance Gene Mutagenesis Mutation Nature Pattern Pharmaceutical Preparations Plasmids Property Protein Family Proteins Public Health Quinolones Resistance Role SOS Response Scanning Shewanella Shock Son of Sevenless Proteins Stenotrophomonas maltophilia Stress Structure Structure-Activity Relationship Surface Plasmon Resonance System Targeted Toxins Testing Titrations Topoisomerase Toxic effect Ultraviolet Rays Vibrio Work aminoglycoside 6&apos -N-acetyltransferase antimicrobial antimicrobial drug bacterial resistance clinically significant deletion analysis efflux pump member microcin mutant overexpression pathogen physical property protein protein interaction quinolone resistance resistance mechanism resistance mutation yeast two hybrid system

项目摘要

Project Summary Quinolones have been very useful antimicrobial agents because they are highly potent, active against a wide range of bacteria, and relatively non-toxic. Their broad use, however, has been followed by rising rates of resistance. Quinolone resistance has traditionally been understood to arise either by mutations that alter DNA gyrase and topoisomerase IV, enzymes that are the targets for quinolone action, or by mutations that increase expression of efflux pumps that actively eliminate the agents from the cell. Neither type of resistance has been transmissible since both are due to mutations on the bacterial chromosome. Hence, it came as a surprise when plasmid-mediated quinolone resistance was discovered. Three distinct mechanisms for such resistance are known: target protection by pentapeptide repeat proteins of the QnrA, QnrB, and QnrS families that may act in part as DNA mimics, quinolone inactivation by mutant aminoglycoside 6' N-acetyltransferase [Aac(6')-Ib- cr], and provision of new systems for quinolone efflux. Each mechanism confers low-level resistance but facilitates selection of higher level, clinically significant resistance. Although plasmid-mediated quinolone resistance was discovered only 11 years ago, subsequent studies have shown the genes to be broadly distributed in gram-negative bacteria from around the world and to be typically incorporated into integrons on multiresistance plasmids. This resubmission application builds on our prior studies to obtain a deeper and more detailed understanding of the resistance due to Qnr proteins. Under Specific Aim 1, we propose to identify essential regions and amino acid residues in QnrB1 via alanine-scanning mutagenesis and deletion analysis. Cloned mutant genes will be screened for ability to confer quinolone resistance and to inhibit bacterial growth. Candidate mutant proteins will be overexpressed, purified, and tested for protection and inhibition of purified gyrase and ability to block DNA binding to gyrase. Under Specific Aim 2, we propose to evaluate the native functions of qnrA, qnrB, and qnrS. We have found a LexA recognition sequence upstream from plasmid-mediated qnrB alleles and have shown that qnrB expression is under SOS control. In Shewanella algae, a reservoir of qnrA, we have further found cold shock to trigger qnrA expression, and we propose to test further conditions of expression in S. algae, Vibrio splendidus, a reservoir of qnrS-like genes, Stenotrophomonas maltophilia, a reservoir of qnrB-like genes, and we will determine the effect of quinolones and other DNA damaging agents, such as ultraviolet light (as well as other conditions of environmental stress) on qnr expression. We will also directly test the hypothesis that Qnr proteins protect against the natural gyrase-targeting toxin microcin B17. In addition we will screen for proteins other than gyrase that interact with Qnr by use of bacterial and yeast two-hybrid systems. Under Specific Aim 3, we propose to explore Qnr/gyrase interaction as revealed by isothermal titration calorimetry or surface plasmon resonance and by x- ray crystallography.

项目概要喹诺酮类药物是非常有用的抗菌剂，因为它们非常有效，对广泛的细菌有活性。细菌范围广，且相对无毒。然而，随着它们的广泛使用，其使用率也随之上升。反抗。传统上认为喹诺酮耐药性是由改变 DNA 的突变引起的旋转酶和拓扑异构酶 IV，这些酶是喹诺酮作用的靶标，或者通过突变增加外排泵的表达，主动消除细胞中的药剂。这两种类型的抵抗都没有被都具有传染性，因为两者都是由于细菌染色体上的突变造成的。于是乎，就让人感到意外了当发现质粒介导的喹诺酮耐药性时。这种抵抗的三种不同机制已知：QnrA、QnrB 和 QnrS 家族五肽重复蛋白的靶标保护可能部分作为 DNA 模拟物，突变型氨基糖苷 6' N-乙酰转移酶 [Aac(6')-Ib- 使喹诺酮失活 cr]，并提供新的喹诺酮流出系统。每种机制都会产生低水平的阻力，但有助于选择更高水平的、具有临床意义的耐药性。尽管质粒介导的喹诺酮耐药性仅在 11 年前被发现，随后的研究表明该基因广泛存在分布于世界各地的革兰氏阴性细菌中，通常被整合到整合子中多重抗性质粒。此重新提交申请以我们之前的研究为基础，以获得更深入和更深入的研究更详细地了解 Qnr 蛋白引起的耐药性。在具体目标 1 下，我们建议通过丙氨酸扫描诱变和缺失鉴定 QnrB1 中的必需区域和氨基酸残基分析。将筛选克隆的突变基因以赋予喹诺酮抗性并抑制细菌生长。候选突变蛋白将被过表达、纯化并测试其保护作用和抑制纯化的促旋酶和阻断 DNA 与促旋酶结合的能力。在具体目标 2 下，我们建议评估 qnrA、qnrB 和 qnrS 的本机函数。我们在上游发现了LexA识别序列来自质粒介导的 qnrB 等位基因，并表明 qnrB 表达受到 SOS 控制。在希瓦氏藻是qnrA的储存库，我们进一步发现冷休克可以触发qnrA表达，并且我们建议进一步测试 S. algae、Vibrio splendidus（qnrS 样基因库）中的表达条件，嗜麦芽寡养单胞菌，qnrB 样基因的储存库，我们将确定喹诺酮类药物的作用和其他 DNA 损伤剂，例如紫外线（以及其他环境压力条件）关于 qnr 表达式。我们还将直接检验 Qnr 蛋白可以防止自然环境侵害的假设。旋转酶靶向毒素小菌素 B17。此外，我们将筛选除旋转酶以外的与以下物质相互作用的蛋白质： Qnr 使用细菌和酵母两种杂交系统。在具体目标 3 下，我们建议探索通过等温滴定量热法或表面等离子体共振以及 x-揭示的 Qnr/旋转酶相互作用射线晶体学。