Plasmid-mediated Quinolone resistance

描述（由申请人提供）：喹诺酮类药物是非常有用的抗菌剂，因为它们非常有效，对各种细菌都有活性，并且相对无毒。然而，随着它们的广泛使用，抗药性也在上升。喹诺酮耐药性传统上被认为是通过改变DNA促旋酶和拓扑异构酶IV（喹诺酮作用的靶酶）的突变或通过增加主动从细胞中消除药剂的外排泵的表达的突变而产生的。这两种类型的耐药性都不是可传播的，因为两者都是由于细菌染色体上的突变。因此，当质粒介导的喹诺酮耐药性被发现时，这是一个惊喜。这种耐药性的三种不同机制是已知的：通过QnrA、QnrB和QnrS家族的五肽重复蛋白的靶保护，其可以部分地作为DNA模拟物，通过突变氨基糖苷6 'N-乙酰转移酶[Aac（6'）-Ib-cr]的喹诺酮失活，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。虽然质粒介导的喹诺酮耐药仅在11年前被发现，但随后的研究表明，这些基因广泛分布于世界各地的革兰氏阴性细菌中，并且通常被整合到多重耐药质粒上的整合子中。此次重新提交申请以我们之前的研究为基础，以更深入、更详细地了解Qnr蛋白引起的耐药性。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鉴定QnrB1中的必需区域和氨基酸残基。将筛选克隆的突变基因赋予喹诺酮抗性和抑制细菌生长的能力。候选突变蛋白将过表达、纯化，并测试纯化促旋酶的保护和抑制以及阻断DNA与促旋酶结合的能力。在具体目标2下，我们建议评估qnrA、qnrB和qnrS的原生功能。我们已经发现了一个莱克萨识别序列上游质粒介导的qnrB等位基因，并已表明，qnrB表达SOS控制。在希瓦氏藻（Shewanella algae）中，我们已经进一步发现冷休克触发qnrA表达，我们建议测试在S.藻类、灿烂弧菌（Vibrio splendidus）、嗜麦芽寡养单胞菌（Stenotrophomonas maltophilia）、qnrB样基因的储存库，并且我们将确定喹诺酮类和其它DNA损伤剂（例如紫外线（以及其它环境应激条件））对qnr表达的影响。我们还将直接测试Qnr蛋白质保护免受天然gyrase靶向毒素microcin B17的假设。此外，我们还将通过细菌和酵母双杂交系统筛选与Qnr相互作用的促旋酶以外的蛋白质。在具体目标3下，我们建议探索Qnr/促旋酶相互作用，如等温滴定量热法或表面等离子体共振和X射线晶体学所揭示的。公共卫生相关性：喹诺酮类是广泛使用的抗菌药物，但已受到细菌耐药性的影响，而细菌耐药性最初被认为仅发生在染色体突变中。质粒编码的可转移耐药性现已被证明已经出现并传播到许多革兰氏阴性人类病原体，并且具有多种机制，显然是选择与喹诺酮靶酶拓扑异构酶相互作用的染色体蛋白。因此，了解这些耐药机制以及编码它们的基因如何被动员和修饰以赋予耐药性对于公共卫生和了解细菌适应策略的范围具有重要意义。

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.