QUINOLONE RESISTANCE MECHANISMS IN STAPHYLOCOCCUS AUREUS

描述（改编自申请人摘要）：PI感兴趣 在研究葡萄球菌属的氟喹诺酮耐药性方面， 三 原因 首先，葡萄球菌是重要的病原体， 对氟喹诺酮类和其他抗生素的耐药性更强。 理解 喹诺酮类耐药的机制，这似乎是不同的， 革兰氏阴性菌中所见的，因此是一个重要的目标。 一 第二个原因是拓扑异构酶IV似乎是主要的氟喹诺酮 革兰氏阳性细菌中的靶点，而不是DNA旋转酶，这使得 有可能研究拓扑异构酶的功能和调节 这在革兰氏阴性菌中是不可能的。 最后，PI是 对多药外排转运蛋白的机制和调节感兴趣 这提供了另一种氟喹诺酮耐药性机制 拓扑异构酶本身的改变。 更具体地说，PI建议 以下几点：1. 为了确定两种拓扑异构酶IV的突变 亚基ParC和ParD使拓扑异构酶对 氟喹诺酮作用。 为此，PI将纯化和表征 突变的拓扑异构酶，以确定它们与抗生素的结合是否 降低或影响酶的催化活性。 成果 分析不仅应该指出阻力机制是如何工作的， 喹诺酮类是否通过降低酶活性或更间接地通过 稳定酶-DNA复合物。 2. 确定拓扑异构酶IV相对于DNA的位置 复制复合体 拓扑异构酶IV被认为是通过脱连环化的 新复制的子染色体，以允许分离。 氟喹诺酮类药物可用于捕获酶-DNA复合物，从而确定 拓扑异构酶起作用的位点。 topo IV优先使用的网站 将被识别。 PI还将确定抗生素 停止新的DNA合成，这表明topo IV是否在附近或附近起作用。 远离复制复合体。 私家侦探已经分离出一种新型的 他希望这种产生耐药性的突变可以揭示 Topo IV参与DNA复制。 这些变种人将会 详细描述。 3. 表征诺拉A的调节和功能， 泵介导氟喹诺酮耐药性。 基因融合将用于 以下表达和各种已知的全球监管机构的影响将是 测定 将定位诺拉A的启动子。 诺拉功能将是 通过纯化诺拉蛋白并将其掺入脂质体中来评估。 4. 表征影响氟喹诺酮类药物演变的因素 阻力 人们注意到，耐甲氧西林S.金黄色葡萄球菌菌株 对氟喹诺酮类药物产生耐药性的可能性远远大于 甲氧西林敏感菌株。 私家侦探有证据表明 可能是由于甲氧西林耐药基因mecA与gyrA的连锁 已经开始在突变途径上检测的基因 阻力 PI还将确定是否诱导 在MSSA菌株中，喹诺酮类药物引起的纤连蛋白结合蛋白 对喹诺酮类耐药的MSSA有扩散的趋势， 与MRSA相比，

DESCRIPTION (Adapted from the applicant's abstract): The PI is interested in studying fluoroquinolone resistance in Staphylococcus spp. for three reasons. First, staphylococci are important pathogens and are growing more and more resistant to fluoroquinolones and other antibiotics. Understanding the mechanism of quinolone resistance, which appears to be different from that seen in the gram-negative bacteria, is thus an important goal. A second reason is that topoisomerase IV seems to be the main fluoroquinolone target in the gram positive bacteria rather than DNA gyrase, and this makes it possible to investigate the function and regulation of topoisomerases in ways not possible in the gram-negative bacteria. Finally, the PI is interested in the mechanism and regulation of multidrug efflux transporters that provide yet another mechanism of fluoroquinolone resistance in addition to alterations in topoisomerase itself. More specifically, the PI proposes the following: 1. To determine how mutations in the two topoisomerase IV subunits, ParC and ParD, make the topoisomerase less susceptible to fluoroquinolone action. To this end, the PI will purify and characterize mutant topoisomerases to determine if their binding of the antibiotic is reduced or affect the catalytic activity of the enzyme. Results of this analysis should indicate not only how the resistance mechanism works but whether quinolones act by reducing enzyme activity or more indirectly by stabilizing enzyme-DNA complexes. 2. To determine the location of topoisomerase IV relative to the DNA replication complex. Topoisomerase IV is thought to act by decatenation of newly replicated daughter chromosomes to allow segregation. Fluoroquinolones can be used to trap enzyme-DNA complexes and thus determine the sites where topoisomerase acts. Sites preferentially used by topo IV will be identified. The PI will also determine how rapidly the antibiotic stops new DNA synthesis, an indication of whether the topo IV acts near or far from the replication complex. The PI has isolated a new type of resistance-producing mutation that he hopes may shed light on the involvement of topo IV in DNA replication. These mutants will be characterized in detail. 3. To characterize the regulation and function of NorA, a multidrug efflux pump that mediates fluoroquinolone resistance. Gene fusions will be used to follow expression and the effect of various known global regulators will be determined. The promoter of norA will be located. NorA function will be assessed by purifying NorA protein and incorporating it in liposomes. 4. To characterize factors affecting the evolution of fluoroquinolone resistance. It has been noted that methicillin-resistant S. aureus strains are far more likely to become resistant to fluoroquinolones than methicillin-sensitive strains. The PI has evidence suggesting that this might be due to linkage of the methicillin resistance gene, mecA, to gyrA genes that have already started on the mutation pathway to detectable resistance. The PI will also determine whether the induction of fibronectin-binding proteins by quinolones in MSSA strains is responsible for the tendency of the MSSA that become quinolone resistant to spread clonally, in contrast to MRSA.