Plasmid-mediated Quinolone Resistance

质粒介导的喹诺酮类耐药

• 批准号: 8695968
• 负责人: David C Hooper
• 金额: $ 41.13万
• 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 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695968
• 关键词: Address; Affect; Alanine; Algae; Antibiotic Resistance; Bacteria; Bacterial Chromosomes; Binding; Biological Models; Cells; Chimeric Proteins; Chromosome Deletion; Chromosomes; Ciprofloxacin; Citrobacter; DNA; DNA Gyrase; DNA Topoisomerase IV; DNA-Binding Proteins; Deletion Mutation; Dissection; Elements; Enzyme Inhibition; Enzymes; Escherichia coli; Family; Fluoroquinolones; Gene Expression; Gene Library; Gene Proteins; Genes; Genome; Gram-Negative Bacteria; Growth; Habitats; Homologous Gene; Human; Hybrids; Integrons; Knock-out; LacZ Genes; Libraries; Link; Mass Spectrum Analysis; Measures; Mediating; Medical; Mobile Genetic Elements; Multi-Drug Resistance; Multidrug Resistance Gene; Mutation; Nature; Organism; Pathway interactions; Pharmaceutical Preparations; Physiological; Plasmids; Positioning Attribute; Predisposition; Principal Investigator; Production; Protein Family; Proteins; Public Health; Quinolones; Regulation; Regulatory Element; Regulatory Pathway; Reporter; Resistance; Shewanella; Site; Son of Sevenless Proteins; Stress; Structure; Structure-Activity Relationship; Subgroup; System; Testing; Topoisomerase; Toxic effect; Toxin; Vibrio; Work; aminoglycoside 6' -N-acetyltransferase; antimicrobial; antimicrobial drug; bacterial resistance; base; clinically significant; crosslink; efflux pump; in vivo; mutant; novel; pathogen; programs; protein protein interaction; public health relevance; quinolone resistance; research study; resistance mechanism; resistance mutation; three dimensional structure

DESCRIPTION (provided by applicant): Fluoroquinolones, such as ciprofloxacin, 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 in 1998. Three distinct mechanisms for such resistance are known: target protection by pentapeptide repeat proteins of the Qnr family that bind gyrase and may act in part as DNA mimics, quinolone inactivation by a 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. The Qnr family is largest group with QnrA, QnrB, QnrS, QnrC, and QnrD subgroups and is now distributed worldwide with the qnr genes generally present within integrons on multidrug resistance plasmids. This renewal 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, based on our mutant Qnr studies we propose to perform site-specific cross linking of key residues of Qnr B with gyrase and analysis of sites of linkage by mass spectrometry. Bacterial 2-hybrid studies will also be used to evaluate binding of specific QnrB and gyrase mutants for their interactions in intact cells. Under Specific Aim 2, we propose to evaluate the native functions of qnr genes and a novel pathway of regulation of qnrS expression. Having excluded Qnr protection of natural gyrase toxins CcdB and ParE, we will next evaluate its protection from natural gyrase toxin MccB17 using plasmid constructs with graded expression of toxin and Qnr. We will also construct deletions of the native homologs qnrA and qnrS in Shewanella algae and Vibrio splendidus, the respective reservoir organisms and test for differences in quinolone susceptibility and growth under environmental conditions relevant for their native habitats. We will also construct a qnrS-lacZ transcriptional fusion to screen an E. coli transposon mutant library for genes necessary for the SOS-independent induction of qnrS by ciprofloxacin. Under Specific Aim 3, we propose to assess Qnr interactions with other proteins by testing the effects of qnr on expression of other E. coli genes in a transcripitional microarray with confirmation in the native organisms and by coimmunoprecipitation experiments for direct identification of binding partners with mass spectrometry analysis.

描述（由申请人提供）：氟喹诺酮类药物，例如环丙沙星，一直是非常有用的抗微生物剂，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 1998 年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：Qnr 家族五肽重复蛋白的靶标保护，该蛋白结合旋转酶并可能部分充当 DNA 模拟物、喹诺酮突变型氨基糖苷 6' N-乙酰转移酶 [Aac(6')-Ib-cr] 失活，并为喹诺酮流出提供新系统。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。 Qnr 家族是最大的群体，包括 QnrA、QnrB、QnrS、QnrC 和 QnrD 亚群，目前分布于世界各地，qnr 基因通常存在于多药耐药质粒的整合子中。这一更新应用建立在我们之前的研究基础上，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，基于我们的突变 Qnr 研究，我们建议对 Qnr B 的关键残基与旋转酶进行位点特异性交联，并通过质谱分析连接位点。细菌 2 杂交研究还将用于评估特定 QnrB 和旋转酶突变体的结合，以了解它们在完整细胞中的相互作用。在具体目标 2 下，我们建议评估 qnr 基因的天然功能和 qnrS 表达调节的新途径。排除了天然促旋酶毒素 CcdB 和 ParE 的 Qnr 保护后，我们接下来将使用具有毒素和 Qnr 分级表达的质粒构建体来评估其对天然促旋酶毒素 McB17 的保护。我们还将在希瓦氏藻和灿烂弧菌（各自的储存生物）中构建天然同源物 qnrA 和 qnrS 的缺失，并测试在与其天然栖息地相关的环境条件下喹诺酮敏感性和生长的差异。我们还将构建 qnrS-lacZ 转录融合体，以筛选大肠杆菌转座子突变体库，以寻找环丙沙星独立于 SOS 诱导 qnrS 所需的基因。在具体目标 3 下，我们建议通过测试 qnr 对转录微阵列中其他大肠杆菌基因表达的影响来评估 Qnr 与其他蛋白质的相互作用，并在天然生物体中进行确认，并通过共免疫沉淀实验直接鉴定具有质量的结合伴侣光谱分析。