Plasmid-mediated Quinolone resistance

质粒介导的喹诺酮类耐药

• 批准号: 8099517
• 负责人: David C Hooper
• 金额: $ 41.09万
• 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/8099517
• 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; Health; 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; 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）：奎诺酮是非常有用的抗菌剂，因为它们具有高效，对广泛的细菌且相对无毒。然而，它们的广泛使用之后，阻力率上升。传统上，奎诺酮的抗性是通过改变DNA陀螺酶和拓扑异构酶IV的突变，即是喹诺酮作用的靶标的突变，或者是通过增加积极消除剂从细胞中消除剂的外排泵表达的突变。两种类型的耐药性均可传播，因为两者都是由于细菌染色体上的突变引起的。因此，当发现质粒介导的喹诺酮耐药性时，这令人惊讶。已知这种抗性的三种不同的机制：QnRA，QNRB和QNRS家族的五肽重复蛋白的靶标的可能部分用作DNA模拟物，突变胺氨基糖苷6'n-乙酰基转移酶[AAC（6'''''aac（6''） - ib- cr- corsoys for Quiner and Systems for New for Quine and Quins和QuinsOrys Quinolone灭活。每种机制都赋予低级电阻，但有助于选择更高水平，临床上显着的阻力。尽管仅11年前才发现了质粒介导的喹诺酮抗性，但随后的研究表明，这些基因将在来自世界各地的革兰氏阴性细菌中广泛分布，并通常在多余质粒上掺入整合剂中。此重新提交应用于我们先前的研究，以获得对QNR蛋白引起的抗性的更深入，更详细的了解。在特定目标1下，我们建议通过丙氨酸扫描诱变和缺失分析来识别QNRB1中的必要区域和氨基酸残基。克隆的突变基因将被筛选，以赋予喹诺酮耐药性和抑制细菌生长的能力。候选突变蛋白将过表达，纯化和测试，以保护和抑制纯化的回旋酶以及阻断DNA与回旋酶结合的能力。在特定目标2下，我们建议评估QNRA，QNRB和QNR的天然功能。我们已经从质粒介导的QNRB等位基因上游发现了LEXA识别序列，并表明QNRB表达在SOS对照下。 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 QNR表达上的紫外线（以及其他环境应力条件）等药物。我们还将直接检验QNR蛋白保护天然旋转酶靶向毒素微蛋白B17的假设。此外，我们还将通过使用细菌和酵母两杂交系统与QNR相互作用的回旋酶以外的其他蛋白质进行筛选。在特定目标3下，我们建议探索QNR/Gyrase的相互作用，如等温滴定量热法或表面等离子体共振以及X-Ray晶体学所揭示的那样。公共卫生相关性：喹诺酮是广泛使用的抗菌剂，已被细菌耐药性损害，最初被认为仅来自染色体突变。现已证明质粒编码的可转移抗性已经出现并扩散到许多革兰氏阴性的人类病原体上，并具有多种机制，显然选择了与拓扑异构酶相互作用的染色体蛋白，即喹诺酮靶酶。因此，了解这些抗药性机制以及编码它们的基因如何动员和修改以赋予抵抗力对于公共卫生和理解细菌适应策略的范围至关重要。