Plasmid-mediated Quinolone resistance

项目摘要 喹诺酮类药物一直是非常有用的抗菌剂，因为它们高度有效，对广泛的 细菌种类繁多，且相对无毒。然而，随着它们的广泛使用， 抵抗。传统上认为，喹诺酮类耐药是由改变dna的突变引起的。 旋转酶和拓扑异构酶IV，这两种酶是喹诺酮类药物作用的靶标，或者是由增加的突变引起的 外排泵的表达能主动地将这些毒剂从细胞中清除。这两种类型的抵抗都没有 可传播，因为两者都是由于细菌染色体上的突变造成的。因此，这是一个惊喜。 当发现质粒介导的喹诺酮类耐药时。产生这种抗药性的三种不同机制 已知：QnrA、QnrB和QnrS家族的五肽重复蛋白可能具有靶向保护作用 突变的氨基糖苷6‘N-乙酰转移酶[AAc(6’)-Ib-]对喹诺酮类药物的灭活作用 Cr]，并提供新的喹诺酮类外排系统。每种机制都会产生低水平的抵抗力，但 有助于选择更高水平的、临床上有意义的耐药性。虽然质粒介导的喹诺酮类药物 抗药性是11年前才发现的，随后的研究表明，这些基因广泛存在 分布在来自世界各地的革兰氏阴性细菌中，通常被整合到 多耐药质粒。这份重新提交的申请建立在我们之前的研究基础上，以获得更深层次的 更详细地了解QnR蛋白引起的抗性。在具体目标1下，我们建议 丙氨酸扫描突变和缺失鉴定QnrB1的必需区域和氨基酸残基 分析。克隆的突变基因将被筛选出赋予喹诺酮类药物耐药性和抑制其耐药性的能力。 细菌生长。候选突变蛋白将被过度表达、纯化并进行保护和测试 抑制纯化的旋转酶和阻断DNA与旋转酶的结合。在具体目标2下，我们建议 评估qnrA、qnrB和qnrS的本机功能。我们在上游发现了一个词汇识别序列 研究表明，qnrB基因的表达受SOS的控制。在……里面 希瓦氏藻，qnrA的储存库，我们进一步发现冷休克可以触发qnrA的表达，我们 建议进一步测试藻类弧菌的表达条件，该弧菌是qnrS样基因的储存库， 嗜麦芽窄食单胞菌，qnrB样基因的储存库，我们将确定喹诺酮类药物的作用 以及其他DNA损伤剂，如紫外线(以及其他环境压力条件) 关于qnr表达。我们还将直接测试QnR蛋白对天然 旋转酶靶向毒素微球素B17。此外，我们还将筛选除旋转酶以外的与 利用细菌和酵母双杂交系统进行QnR。在具体目标3下，我们建议探讨 QnR/旋转酶相互作用的等温滴定热法或表面等离子体共振法以及X-射线衍射法。 射线结晶学。

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.