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药物，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，当质粒介导的喹诺酮类药物耐药性被发现时，人们感到非常惊讶。这种耐药的三种不同机制是已知的：QnrA、QnrB和QnrS家族的五肽重复蛋白（可能部分作为DNA模拟物）的靶标保护，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。虽然质粒介导的喹诺酮类药物耐药性仅在11年前被发现，但随后的研究表明，这些基因广泛分布在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。这项重新提交的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Specific Aim 1下，我们建议通过丙氨酸扫描诱变和缺失分析来确定QnrB1的必要区域和氨基酸残基。克隆的突变基因将被筛选是否具有喹诺酮类药物耐药性和抑制细菌生长的能力。候选突变蛋白将被过表达、纯化，并测试其对纯化的gyrase的保护和抑制作用以及阻断DNA与gyrase结合的能力。在具体目标2中，我们建议评估qnrA， qnrB和qnrS的天然功能。我们在质粒介导的qnrB等位基因上游发现了一个LexA识别序列，并表明qnrB的表达受SOS控制。在qnrA的储存库Shewanella藻类中，我们进一步发现了冷休克触发qnrA的表达，我们建议进一步测试S. algae、spldibrio spldius （qnrs样基因储存库）、Stenotrophomonas maltopia （qnrb样基因储存库）的表达条件，我们将确定喹诺酮类药物和其他DNA损伤剂，如紫外线（以及其他环境胁迫条件）对qnr表达的影响。我们还将直接验证Qnr蛋白对天然靶向gyase毒素microcin B17的保护作用。此外，我们将使用细菌和酵母双杂交系统筛选除gyrase外与Qnr相互作用的蛋白质。在具体目标3中，我们建议通过等温滴定量热法或表面等离子体共振和x射线晶体学来探索Qnr/gyrase的相互作用。公共卫生相关性：喹诺酮类药物是广泛使用的抗微生物药物，但已受到细菌耐药性的损害，最初认为这种耐药性仅由染色体突变引起。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中，并且具有多种机制，显然选择了与拓扑异构酶（喹诺酮靶酶）相互作用的染色体蛋白。因此，了解这些耐药性机制以及编码这些机制的基因如何被调动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.