Plasmid-mediated Quinolone resistance

质粒介导的喹诺酮类耐药

• 批准号: 7736766
• 负责人: David C Hooper
• 金额: $ 47.21万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 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/7736766
• 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' -N-acetyltransferase; antimicrobial; antimicrobial drug; bacterial resistance; clinically significant; deletion analysis; efflux pump; member; microcin; mutant; overexpression; pathogen; physical property; protein protein interaction; public health relevance; quinolone resistance; resistance mechanism; resistance mutation; yeast two hybrid system

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Quinolones are widely used antimicrobial agents that have been compromised by bacterial resistance, which was originally thought only to occur from chromosomal mutation. Plasmid-encoded transferable resistance has now been shown to have emerged and spread to many gram-negative human pathogens and to have a diversity of mechanisms, apparently co-opting chromosomal proteins that interact with topoisomerases, the quinolone target enzymes. Thus, understanding of these mechanisms of resistance and how the genes encoding them have been mobilized and modified to confer resistance is of importance for public health and for understanding of the range of bacterial adaptation strategies.

描述（由申请人提供）：喹诺酮类药物一直是非常有用的抗菌剂，因为它们非常有效，对多种细菌具有活性，并且相对无毒。然而，随着它们的广泛使用，抵制率也随之上升。传统上认为喹诺酮耐药性是由改变 DNA 旋转酶和拓扑异构酶 IV（喹诺酮作用靶标酶）的突变引起的，或者是由增加主动消除细胞内药物的外排泵表达的突变引起的。这两种类型的耐药性都不会传播，因为它们都是由细菌染色体突变引起的。因此，当发现质粒介导的喹诺酮耐药性时，人们感到惊讶。已知这种耐药性的三种不同机制：QnrA、QnrB 和 QnrS 家族五肽重复蛋白的靶标保护（可能部分充当 DNA 模拟物）、突变型氨基糖苷 6' N-乙酰转移酶 [Aac(6')- 导致的喹诺酮失活。 Ib-cr]，并提供用于喹诺酮流出的新系统。每种机制都赋予低水平的耐药性，但有利于选择更高水平的、临床上显着的耐药性。尽管质粒介导的喹诺酮耐药性仅在 11 年前被发现，但随后的研究表明这些基因广泛分布于世界各地的革兰氏阴性细菌中，并且通常被整合到多耐药质粒上的整合子中。此次重新提交申请以我们之前的研究为基础，旨在更深入、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分析来鉴定 QnrB1 中的必需区域和氨基酸残基。将筛选克隆的突变基因以赋予喹诺酮抗性和抑制细菌生长的能力。候选突变蛋白将被过表达、纯化，并测试其对纯化促旋酶的保护和抑制以及阻断 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。我们在质粒介导的 qnrB 等位基因上游发现了 LexA 识别序列，并表明 qnrB 表达受到 SOS 控制。在希瓦氏藻（qnrA 的储存库）中，我们进一步发现冷休克可触发 qnrA 表达，并且我们建议测试在 S. algae、灿烂弧菌（qnrS 样基因的储存库）、嗜麦芽窄食单胞菌（qnrA 的储存库）中的进一步表达条件。 qnrB 样基因的影响，我们将确定喹诺酮类药物和其他 DNA 损伤剂的影响，例如紫外线（以及其他环境条件）压力）对 qnr 表达的影响。我们还将直接检验以下假设：Qnr 蛋白可以抵御天然促旋酶靶向毒素小菌素 B17。此外，我们将利用细菌和酵母双杂交系统筛选除旋转酶之外与 Qnr 相互作用的蛋白质。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 射线晶体学揭示的 Qnr/旋转酶相互作用。公共健康相关性：喹诺酮类药物是广泛使用的抗菌药物，但已受到细菌耐药性的影响，最初认为细菌耐药性仅由染色体突变引起。现已证明，质粒编码的可转移抗性已经出现并传播到许多革兰氏阴性人类病原体，并且具有多种机制，显然是选择与拓扑异构酶（喹诺酮靶酶）相互作用的染色体蛋白。因此，了解这些耐药机制以及编码它们的基因如何被动员和修饰以赋予耐药性对于公共卫生和了解细菌适应策略的范围非常重要。