QUINOLONE RESISTANCE MECHANISMS IN STAPHYLOCOCCUS AUREUS

描述(摘自申请人摘要)：金黄色葡萄球菌是一种 重要的病原体，因为它是常见的、剧毒的和容易发展的 抵抗。随着对其他药物的耐药性增加，临床医生 被迫依赖新的药物，如喹诺酮类药物。喹诺酮类药物 抗生素是非常有效的制剂，但出现抗药性 喹诺酮类药物很容易出现。通过了解以下机制： 抗药性，可以设计策略和开发新药来 绕过阻力。基于在大肠杆菌中的研究，有两种常见的机制 已确定的耐喹诺酮类药物有：1)点突变 导致靶酶、DNA旋转酶和 2)减少药物对其目标的渗透，主要是由于 外膜蛋白发生变化。金黄色葡萄球菌耐药机制的研究进展 很可能与大肠杆菌中的不同，因为葡萄球菌 没有限制通透性的外膜。也是单步走 耐药突变体的特征是没有旋转酶突变 存在一个新的基因座flqA，与gyrA或gyrB没有遗传连锁关系 牵涉其中。FlqA还确定旋转酶突变是否包括 11月(可能与gyrB相同，并决定新生物 抗药性)表达或不表达。这项研究的主旨是 鉴定由flqA编码的基因产物。大量数据表明 该flqA编码一个拓扑异构酶IV蛋白。胡珀博士已经确定 另外两个基因座flqB和flqC参与了抗性的表达。 FlqB是NorA基因上游的一个点突变，NorA是一种编码外排的基因 泵，其过度表达导致喹诺酮类耐药 从细胞中抽出更多的喹诺酮类药物。FlqC可能是一个 GyrA的抗性等位基因。 有四个目标：1)flqA基因座的特征，其 与拓扑异构酶IV的关系及其与flqC基因座的相互作用。 基于数据表明flqA类似于gyrA或gyrB，并且 与flqA+、lambda克隆相比，flqA中存在突变 包含这些序列的序列将首先亚克隆到E.Coli(和 测序)，然后进入金黄色葡萄球菌，在后一种情况下通过单拷贝 整合载体或在一个质粒上，以确定对表型的影响。 拓扑异构酶基因将在大肠杆菌中表达并纯化用于 生化研究。FlqA基因突变对NOV和FlqC的影响 通过引入flqA突变体和flqA+在 转化金黄色葡萄球菌。FlqC对flqA表达的影响 用含有β-内毒素的质粒转化金黄色葡萄球菌进行检测 内酰胺酶与flqA启动子的融合及β-内酰胺酶的检测 活动。2)鉴定flqC为gyrA等位基因，nov为a. GyrB等位基因。从gyrA和gyrB中扩增出的DNA将被测序 鉴定与flqC和nov相关的突变。野生型Gyra和 克隆在温度敏感质粒上的gyrB将用于转化 金黄色葡萄球菌将整合到宿主染色体中以“剔除” FlqC和nov的抗性，从而建立了它们与gyrA的同一性 和gyrB。3)调节函数和正规函数的刻画 关于诺拉的。正常NorA启动子(即flqB+)和flqB突变 每一个都将被克隆到Blaz的上游，而各自的质粒将 用于转化flqB和flqB+金黄色葡萄球菌。美国政府的影响 将通过测量来检测NorA转录上的启动子突变 β-内酰胺酶活力。将进行类似的实验，以 研究flqB与其他抗性基因座的相互作用 环境因素对基因表达的影响。多克隆抗血清 将针对Nora准备用于免疫印迹分析 临床分离株产生诺拉的特征。研究以确定 Nora是否是必需的将使用等位基因替代进行研究 或温度敏感的质粒组分。4)定义决定因素 金黄色葡萄球菌临床分离株的耐药性和敏感性。 将在临床分离株中寻找flqA和flqC的存在 用flqA+和flqC+质粒进行互补实验。诺拉 免疫印迹将比较临床分离株之间的表达 实验。

DESCRIPTION (Adapted from applicant's abstract): S. aureus is an important pathogen because it is common, virulent, and prone to develop resistance. As resistance to other drugs increases, clinicians are forced to rely on new agents, such as the quinolones. Quinolone antibiotics are very effective agents, but emergence of resistance to quinolones occurs readily. By understanding the mechanisms of resistance, strategies may be devised and new drugs developed to circumvent resistance. Based on work in E. coli, two general mechanisms of resistance to quinolones have been identified: 1) point mutations leading to structural alterations in the target enzyme, DNA gyrase and 2) reduced permeation of drug to its target, primarily as a result of outer membrane protein changes. Mechanisms of resistance in S. aureus are likely to be different from those in E. coli because staphylococci have no outer membrane to limit permeability. Also single step resistant mutants characteristically do not have gyrase mutations present and a novel locus, flqA, not genetically linked to gyrA or gyrB is involved. flqA also determines whether gyrase mutations, including nov (probably the same as gyrB and which determines novobiocin resistance) are expressed or not. The main thrust of the research is to identify the gene product encoded by flqA. Considerable data indicate that flqA encodes a topoisomerase IV protein. Dr. Hooper has identified two other loci, flqB and flqC, involved in expression of resistance. flqB is a point mutation upstream of norA, a gene encoding an efflux pump, over-expression of which leads to quinolone resistance by virtue of increased pumping of quinolone out of the cell. flqC is probably a resistant allele of gyrA. There are four aims: 1) Characterization of the flqA locus, its relationship to topoisomerase IV, and its interactions with flqC locus. Based on data indicating that flqA is gyrA- or gyrB-like and that mutations are present in flqA compared to flqA+, lambda clones containing these sequences will be subcloned first into E. coli (and sequenced) then into S. aureus, in the latter case via a single copy integration vector or on a plasmid, to determine effects on phenotype. Topoisomerase genes will be expressed in E. coli and purified for biochemical studies. The effects of flqA mutations on nov and flqC expression will be examined by introducing flqA mutants and flqA+ on a plasmid into S. aureus. The effect of flqC on flqA expression will be examined by transforming S. aureus with a plasmid containing beta- lactamase fused to the flqA promoter, and assaying for beta-lactamase activity. 2) Identification of flqC as an allele of gyrA and nov as an allele of gyrB. PCR amplified DNA from gyrA and gyrB will be sequenced to identify mutations associated with flqC and nov. Wild-type gyrA and gyrB cloned on a temperature sensitive plasmid will be used to transform S. aureus to integrate into the host chromosome to "cross out" resistance of flqC and nov, thereby establishing their identity with gyrA and gyrB. 3) Characterization of the regulation and normal functions of NorA. The normal norA promoter (i.e., flqB+) and the flqB mutation each will be cloned upstream of blaZ and the respective plasmids will be used to transform flqB and flqB+ S.aureus. The effect of the promoter mutation on norA transcription will be assayed by measuring beta-lactamase activity. Similar experiments will be performed to investigate the interaction of flqB with other resistance loci and the effects of environmental factors on expression. Polyclonal antiserum will be prepared against NorA for use in immunoblotting assays to characterize NorA production in clinical isolates. Studies to determine whether norA is essential will be conducted using allelic replacement or temperature sensitive plasmid constructs. 4) Defining determinants of resistance and susceptibility in clinical isolates of S. aureus. Presence of flqA and flqC in clinical isolates will be sought by complementation experiments using flqA+ and flqC+ plasmids. norA expression will be compared among clinical isolates in immunoblotting experiments.