Plasmid-mediated Quinolone resistance

项目摘要 喹诺酮是非常有用的抗微生物剂，因为它们是高度有效的，对广泛的 范围内的细菌，并相对无毒。然而，随着它们的广泛使用， 阻力传统上认为喹诺酮类药物耐药性是由改变DNA的突变引起的， 促旋酶和拓扑异构酶IV，是喹诺酮作用的靶点的酶，或通过增加的突变 表达主动从细胞中消除试剂的外排泵。这两种抵抗都没有 因为两者都是由于细菌染色体上的突变而导致的。因此， 当质粒介导的喹诺酮耐药性被发现时。这种抵抗的三种不同机制 已知：通过QnrA、QnrB和QnrS家族的五肽重复蛋白的靶向保护， 部分作为DNA模拟物，通过突变氨基糖苷6'N-乙酰转移酶[Aac（6'）-Ib- CR]，并提供喹诺酮外排的新系统。每种机制都能产生低水平的抵抗力， 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 耐药性仅在11年前被发现，随后的研究表明，这些基因广泛存在于 分布在来自世界各地的革兰氏阴性细菌中，并且通常被整合到整合子中。 多抗性质粒这个重新提交的应用程序建立在我们以前的研究，以获得更深入的， 更详细地了解由于Qnr蛋白的抗性。在具体目标1下，我们建议 通过丙氨酸扫描突变和缺失鉴定QnrB 1中必需区域和氨基酸残基 分析.将筛选克隆的突变基因赋予喹诺酮耐药性和抑制 细菌生长候选突变蛋白将被过表达、纯化并测试其保护性， 抑制纯化的促旋酶和阻断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的本机功能。我们发现了一个莱克萨识别序列上游 从质粒介导的qnrB等位基因，并已表明qnrB的表达是在SOS控制。在 希瓦氏藻，qnrA的水库，我们已经进一步发现冷休克触发qnrA的表达，我们 建议进一步测试S中表达的条件。藻类，灿烂弧菌，一个类似qnrS基因的水库， 嗜麦芽窄食单胞菌，qnrB样基因的水库，我们将确定喹诺酮类药物的效果 和其他DNA损伤剂，如紫外线（以及其他环境压力条件） qnr表达。我们还将直接测试Qnr蛋白质保护免受自然环境影响的假设。 靶向脑回酶的毒素小菌素B17。此外，我们还将筛选除促旋酶以外的蛋白质， 利用细菌和酵母双杂交系统研究Qnr。在具体目标3下，我们建议探讨 用等温滴定量热法或表面等离子体共振法和X- 射线晶体学

Project Summary 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.