Plasmid-mediated Quinolone Resistance

描述（由申请人提供）：氟喹诺酮类药物，如环丙沙星，是非常有用的抗菌剂，因为它们具有很强的效力，对多种细菌都有活性，而且相对无毒。然而，随着它们的广泛使用，耐药性也在上升。喹诺酮类药物耐药传统上被认为是由改变DNA旋切酶和拓扑异构酶IV的突变引起的，这些酶是喹诺酮类药物作用的目标，或者是由增加外排泵表达的突变引起的，外排泵主动地将药物从细胞中清除。这两种抗性都不具有传染性，因为它们都是由于细菌染色体上的突变。因此，当1998年发现质粒介导的喹诺酮类药物耐药性时，人们感到惊讶。这种抗性的三种不同机制是已知的：Qnr家族的五肽重复蛋白结合旋转酶并可能部分起DNA模拟物的作用，喹诺酮类药物被突变氨基糖苷6' n -乙酰转移酶[Aac(6')-Ib-cr]失活，以及提供喹诺酮类药物外排的新系统。每种机制都产生低水平的耐药，但有利于选择更高水平的临床显著耐药。Qnr家族是QnrA、QnrB、QnrS、QnrC和QnrD亚群中最大的一类，目前分布在世界各地，Qnr基因通常存在于多药耐药质粒的整合子中。这项更新应用建立在我们之前的研究基础上，以获得对Qnr蛋白抗性的更深入和更详细的了解。在Specific Aim 1下，基于我们的突变Qnr研究，我们建议用gyrase对Qnr B的关键残基进行位点特异性交联，并通过质谱分析连锁位点。细菌2杂交研究也将用于评估特异性QnrB和gyrase突变体在完整细胞中的相互作用。在Specific Aim 2下，我们提出评估qnr基因的天然功能和qnr表达调控的新途径。在排除了Qnr对天然螺旋酶毒素CcdB和ParE的保护作用后，我们接下来将使用毒素和Qnr分级表达的质粒构建物来评估其对天然螺旋酶毒素MccB17的保护作用。我们还将在希瓦氏菌藻类和脾弧菌中构建原生同源物qnrA和qnrS的缺失，并在与其原生栖息地相关的环境条件下测试喹诺酮类药物的敏感性和生长差异。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，以寻找环丙沙星不依赖于sos诱导qnrS所需的基因。在Specific Aim 3下，我们建议通过转录微阵列测试Qnr对其他大肠杆菌基因表达的影响，并在原生生物中得到证实，以及通过共免疫沉淀实验通过质谱分析直接鉴定结合伙伴，来评估Qnr与其他蛋白质的相互作用。

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.