Plasmid-mediated Quinolone resistance

说明(申请人提供)：喹诺酮类药物一直是非常有用的抗菌剂，因为它们非常有效，对多种细菌都有效，而且相对无毒。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，当发现质粒介导的喹诺酮耐药时，这是令人惊讶的。已知这种耐药性的三种不同机制：QnrA、QnrB和QnrS家族的五肽重复蛋白(部分作为DNA模拟物)的靶向保护，突变的氨基糖苷6‘N-乙酰转移酶[Aac(6’)-Ib-cr]使喹诺酮类药物失活，以及提供新的喹诺酮类药物外排系统。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。虽然质粒介导的喹诺酮类耐药在11年前才被发现，但随后的研究表明，这些基因在世界各地的革兰氏阴性菌中广泛分布，并典型地整合到多重耐药质粒上的整合子中。这种重新提交的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。在特定目标1下，我们建议通过丙氨酸扫描突变和缺失分析来确定QnrB1的必需区域和氨基酸残基。将对克隆的突变基因进行筛选，以确定是否具有对喹诺酮类药物的耐药性和抑制细菌生长的能力。候选突变蛋白将被过度表达、纯化，并测试对纯化的旋转酶的保护和抑制以及阻止DNA与旋转酶的结合的能力。在具体目标2下，我们建议评估qnrA、qnrB和qnrS的天然功能。我们在质粒介导的qnrB等位基因的上游发现了一个LexA识别序列，并表明qnrB的表达受SOS控制。在qnrA的储存库Shewanella藻中，我们进一步发现冷休克可以触发qnrA的表达，我们建议测试进一步在藻类中的表达条件，包括qnrS类基因的储存库--华丽弧菌、嗜麦芽窄食单胞菌--以及qnrB-样基因的储存库--嗜麦芽窄食单胞菌，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。我们还将直接测试QnR蛋白对天然旋转酶靶向毒素microcin B17的保护作用这一假设。此外，我们将使用细菌和酵母双杂交系统筛选与QnR相互作用的旋转酶以外的蛋白质。在特定目标3下，我们建议用等温滴定量热法或表面等离子体共振和X射线结晶学研究QnR/旋转酶的相互作用。与公共卫生相关：喹诺酮类药物是广泛使用的抗菌剂，已受到细菌耐药性的影响，最初认为只有染色体突变才会发生耐药性。现在已经证明，质粒编码的可转移耐药性已经出现并传播到许多革兰氏阴性人类病原体，并具有多种机制，显然是与喹诺酮类靶标酶--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是如何被动员和修饰以赋予耐药性的，对于公共卫生和了解细菌适应策略的范围是重要的。

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.