Plasmid-mediated Quinolone Resistance

描述（由申请人提供）：氟喹诺酮类，如环丙沙星，是非常有用的抗菌剂，因为它们是高效的，对广泛的细菌有活性，并且相对无毒。然而，随着它们的广泛使用，抗药性也在上升。传统上认为喹诺酮类药物耐药是由于DNA促旋酶和拓扑异构酶IV（喹诺酮作用的靶酶）发生突变，或由于增加主动清除细胞中药物的外排泵表达的突变而引起的。这两种类型的耐药性都是不可传播的，因为两者都是由于细菌染色体突变造成的。因此，当1998年发现质粒介导的喹诺酮耐药性时，这是一个惊喜。已知这种耐药的三种不同机制：Qnr家族的五肽重复蛋白结合促旋酶并可部分充当DNA模拟物的靶标保护，喹诺酮通过突变氨基糖苷6'N-乙酰转移酶[Aac（6'）-Ib-cr]失活，以及提供喹诺酮外排的新系统。每种机制都赋予低水平的耐药性，但有助于选择更高水平的临床显著耐药性。Qnr家族是最大的群体，具有QnrA、QnrB、QnrS、QnrC和QnrD亚群，并且现在分布于世界各地，其中qnr基因通常存在于多药耐药质粒上的整合子中。该更新申请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。在特定目标1下，基于我们的突变体Qnr研究，我们建议对Qnr B的关键残基与促旋酶进行位点特异性交联，并通过质谱法分析连接位点。细菌双杂交研究也将用于评价特异性QnrB和促旋酶突变体在完整细胞中相互作用的结合。在特定目标2下，我们建议评估qnr基因的天然功能和qnrS表达调控的新途径。在排除了天然促旋酶毒素CcdB和帕雷的Qnr保护后，我们接下来将使用具有毒素和Qnr的分级表达的质粒构建体评估其对天然促旋酶毒素MccB 17的保护。我们还将构建希瓦氏藻和灿烂弧菌，各自的水库生物体和测试喹诺酮类药物的敏感性和生长的差异，在环境条件下的原生同源qnrA和qnrS的缺失与它们的原生栖息地。我们还将构建qnrS-lacZ转录融合体，以筛选E.大肠杆菌转座子突变体库的基因所必需的SOS非依赖性诱导qnrS环丙沙星。在具体目标3下，我们建议通过测试qnr对其他E.大肠杆菌基因在转录微阵列中与天然生物体中的确认，并通过共免疫沉淀实验与质谱分析直接鉴定结合伴侣。

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.