Quinolone Resistance Mechanisms in Staphylococcus aureus

描述(申请人提供)：金黄色葡萄球菌对喹诺酮类药物的耐药机制。喹诺酮类抗菌药作用于两种拓扑异构酶，广泛用于人类感染。它们对常见的金黄色葡萄球菌感染的使用受到了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。确定这些机制是避免耐药性的战略的关键。该项目的长期目标是使用喹诺酮类药物和耐药突变株作为模型系统，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。具体目的是(1)确定拓扑异构酶IV的新突变在影响耐药性和改变酶功能中的作用。突变的酶将被提纯并研究它们的催化功能、DNA和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)利用野生型和突变酶与DNA和药物形成的复合体的X射线结晶学，确定喹诺酮类化合物与拓扑异构酶IV、DNA和喹诺酮复合体的相互作用部位。目的(3)利用NorA启动子DNA和纯化的NorR蛋白的DNA足迹研究NorR蛋白调节NorA外排泵和可能的其他相关泵表达的机制。我们还将使用DNA微阵列与从NorR和arlS突变株制备的RNA杂交的DNA微阵列，对编码外排泵的基因的表达进行转录图谱分析，这两种突变都会影响NorA的表达。目的(4)是通过纯化除NorR外还与NorA上游结合的28kd蛋白，鉴定编码该基因的基因，并在该基因中产生突变，以确定调控NorA表达的其他因素。目的(5)确定多药耐药泵的互补，并分析它们在脓肿模型中的表达。这项工作将通过分析与已知的MDR泵及其调节器相关的基因，并在金黄色葡萄球菌中选择性克隆和过度表达这些基因来完成。这些基因的整体表达模式将通过DNA微阵列中的转录图谱进行比较，使用的是从大鼠皮下脓肿中存活的细菌制备的RNA和来自体外生长的细菌的RNA。

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.