Quinolone Resistance Mechanisms in Staphylococcus aureus

描述（由申请方提供）：金黄色葡萄球菌中的喹诺酮耐药机制。喹诺酮类抗菌剂作用于两种拓扑异构酶，广泛用于人类感染。其用途为普通S。金黄色葡萄球菌感染受到新出现的耐药性的限制。耐药机制包括拓扑异构酶改变和多药耐药（MDR）外排泵表达增加。明确这些机制是避免耐药性战略的关键。该项目的长期目标是使用喹诺酮和耐药突变体作为模型系统，研究在许多细菌中发现的拓扑异构酶和外排泵的控制和功能。具体目标是（1）确定拓扑异构酶IV中的新突变在影响耐药性和改变酶功能中的作用。突变酶将被纯化并研究其催化功能和DNA与喹诺酮类药物的结合以及喹诺酮类药物诱导的DNA切割的形成。目的（2）用X射线晶体学方法研究喹诺酮与拓扑异构酶IV、DNA和喹诺酮复合物的相互作用位点。目的（3）利用诺拉A启动子DNA和纯化的NorR的DNA足迹法，鉴定NorR蛋白调节诺拉外排泵和可能的其他相关泵表达的机制。我们还将使用DNA微阵列对编码外排泵的基因的表达进行转录谱分析，所述DNA微阵列与从具有norR和arlS突变的菌株制备的RNA杂交，这两种突变均影响诺拉表达。目的（4）是通过纯化除NorR外还结合诺拉上游的28-kd蛋白质，鉴定编码基因，并在该基因中产生突变体，鉴定调节诺拉表达的其他因子。目的（5）鉴定多药耐药泵的补体并分析其在脓肿模型中的表达。这项工作将通过分析与已知MDR泵及其调节因子相关的基因，并选择性地克隆和过表达这些基因来完成。金黄色。将使用从大鼠皮下脓肿中存活的细菌制备的RNA和体外生长的细菌的RNA，通过DNA微阵列中的转录谱比较基因表达的总体模式。

DESCRIPTION (provided by applicant): Quinolone Resistance Mechanisms in Staphylococcus aureus. Quinolone antimicrobials act on two topoisomerases and are used widely in human infections. Their use for common S. aureus infections has been limited by emerging resistance. Resistance mechanisms include altered topoisomerases and increased expression of multidrug resistance (MDR) efflux pumps. Defining these mechanisms is key to strategies to avoid resistance. Long-term objectives of the project are to use quinolones and resistant mutants as a model system to study the control and function of topoisomerases and efflux pumps found in many bacteria. Specific aims are (1) to define the roles of novel mutations in topoisomerase IV in effecting resistance and altering enzyme function. Mutant enzymes will be purified and studied for their catalytic functions and binding of DNA and quinolones as well as formation of quinolone-induced DNA cleavage. Aim (2) is to identify the sites of quinolone interaction with complexes of topoisomerase IV, DNA, and quinolone using x-ray crystallography of wildtype and mutant enzymes complexed with DNA and drug. Aim (3) is to identify the mechanism by which the NorR protein regulates expression of the Nora efflux pump and possibly other related pumps using DNA footprinting with norA promoter DNA and purified NorR. We will also perform transcriptional profiling of the expression of genes encoding efflux pumps with DNA microarrays hybridized with RNA prepared from strains with mutations in norR and arlS, both of which affect norA expression. Aim (4) is to identify additional factors regulating norA expression by purification of a 28-kd protein that in addition to NorR binds upstream of norA, identification of the gene encoding, and generating mutants in this gene. Aim (5) is to identify the complement of multidrug resistance pumps and analyze their expression in an abscess model. This work will be done by analysis of genes related to those of known MDR pumps and their regulators and selected cloning and overexpression of these genes in S. aureus. Overall patterns of expression of the genes will be compared by transcriptional profiling in DNA microarrays using RNA prepared from bacteria surviving in a subcutaneous abscess in rats and RNA from bacteria grown in vitro.