Plasmid-mediated Quinolone Resistance

说明(申请人提供)：氟喹诺酮类药物，如环丙沙星，一直是非常有用的抗菌剂，因为它们高度有效，对多种细菌有效，并且相对无毒。然而，随着它们的广泛使用，抵抗率一直在上升。传统上认为，喹诺酮类药物耐药的原因有两种，一种是改变DNA旋转酶和拓扑异构酶IV的突变，这两种酶是喹诺酮类药物作用的靶点；另一种是增加外排泵表达的突变，从而主动将这些药物从细胞中清除出去。这两种耐药性都不是可传播的，因为这两种耐药性都是由于细菌染色体上的突变造成的。因此，1998年发现质粒介导的喹诺酮类耐药令人惊讶。已知这种耐药性的三种不同机制：结合旋转酶并可能部分作为DNA模拟物的QnR家族五肽重复蛋白的靶向保护，突变的氨基糖苷6‘N-乙酰基转移酶[Aac(6’)-Ib-cr]使喹诺酮类药物失活，以及为喹诺酮类药物外排提供新的系统。每种机制都提供低水平的耐药，但有助于选择更高水平的临床显著耐药。QnR家族是由QnrA、QnrB、QnrS、QnrC和QnrD亚群组成的最大的家族，目前广泛分布于世界各地，qnr基因普遍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，以获得对QnR蛋白引起的抗性的更深入和更详细的了解。在具体目标1下，基于我们对突变的QnR的研究，我们建议将QnR B的关键残基与旋转酶进行位点特异性的交联，并用质谱仪分析连锁的位点。细菌双杂交研究也将用于评估特定的QnrB和旋转酶突变体在完整细胞中的相互作用。在特定的目标2下，我们建议评估qnr基因的天然功能和一种新的qnrS表达调控途径。在排除了天然旋转酶毒素CcdB和pARE对QnR的保护后，我们接下来将使用毒素和QnR分级表达的质粒构建来评估其对天然旋转酶毒素MccB17的保护作用。我们还将构建删除希瓦纳藻和壮丽弧菌中的天然同系物qnrA和qnrS，并测试它们对喹诺酮类药物的敏感性和在与其原生栖息地相关的环境条件下的生长差异。我们还将构建qnrS-lacZ转录融合以筛选大肠杆菌转座子突变文库，以寻找环丙沙星诱导非SOS依赖的qnrS所必需的基因。在特定目标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，并通过免疫共沉淀实验直接用质谱分析鉴定结合伙伴。

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.