Quinolone and multidrug resistance in Staphylococcus aureus

描述(申请人提供)：我们建议研究染色体编码的多药耐药(MDR)外排泵的表达调控及其在金黄色葡萄球菌耐药中的作用，重点是对喹诺酮类药物的耐药性。在之前的工作中，我们发现了由NorA、B和C泵表达增加引起的喹诺酮类耐药性，由Tet38泵引起的四环素耐药性，由ABCA泵引起的(-内酰胺类耐药性)，以及其他由MEPA泵引起的杀菌剂耐药性。我们还发现了Pump表达的直接转录调节因子MGRA和NorG，它们相互作用，具有广泛的影响。有五个具体目标。在目标1中，我们将定义MGRA和NorG在NorA和其他编码外排泵的基因表达中的相互作用。我们特别假设MGRA的磷酸化改变了它与NorG的相互作用，改变了它对Pump基因转录的影响。这项工作将包括通过Northern杂交测量HprK激酶构建突变菌株中NorA的表达，以及测量磷酸化和非磷酸化MGRA的启动子结合。在目标2中，我们将通过从细胞提取液中分离与固定化启动子DNA片段结合的蛋白质，通过质谱学确定它们的身份，并产生具有所鉴定基因突变和过度表达的菌株，从而鉴定NorB、te38和ABCA的直接调控因子。这些菌株将通过Northern杂交和转录图谱来评估编码泵的基因表达的变化。在目标3下，我们将通过NorG突变和过表达的菌株的转录图谱来评估NorG的全球效应。在目标4下，我们将确定在调控网络和环境压力的微阵列分析中过度表达的其他候选外排泵，并确定它们在抗生素耐药性中的作用。在SARA、MGRA和ROT全球调控突变体中，与所列基因不同的特定新候选Pump基因已被证明过表达，并且在小鼠皮下脓肿模型中引起严格反应或生长的条件下。这些候选的Pump基因将被克隆，并将评估它们的过度表达对抗生素和杀生剂敏感性的影响，以补充已知的外排耐药机制。在目标5中，我们将确定在目标4中确定的Norb、teT38和其他外排泵基因表达增加的环境触发因素。我们已经证明Norb和te38在金黄色葡萄球菌脓肿中过表达，这些基因的敲除降低了对脓肿环境的适应性。因此，我们假设泵对于脓肿中的细菌生存是重要的，部分原因是相对地保护了抗菌肽，抗菌肽存在于高浓度。我们将通过Northern印迹检测Pump基因的RNA水平，专门测试低铁条件和高浓度抗菌肽作为Pump表达诱导剂的作用，并在体外确定Pump过表达对抗菌肽抗性的影响。与公共卫生相关的常见致病菌，如金黄色葡萄球菌，在医院和社区的耐药性正在增加。外排泵用于保护细菌免受不利环境的影响。由多药耐药外排泵产生的多药耐药(MDR)和喹诺酮类耐药也会影响药物效用，影响患者对抗生素的反应。因此，确定金黄色葡萄球菌中MDR外排泵的全部组成以及它们的调控方式将为了解葡萄球菌在不同环境中致病的适合性以及优化抗生素的作用和使用提供重要的实用和基本信息。

DESCRIPTION (provided by applicant): We propose to study the regulation of expression of chromosomally encoded multidrug resistance (MDR) efflux pumps and their roles in antimicrobial resistance in Staphylococcus aureus, with a focus on quinolone resistance. In prior work we identified quinolone resistance caused for increased expression of the NorA, B, and C pumps, tetracycline resistance due to the Tet38 pump, (-lactam resistance due to the AbcA pump, and others have identified biocide resistance due to the MepA pump. We have also identified direct transcriptional regulators of pump expression, MgrA and NorG, which interact with each other and have broad effects. There are five Specific Aims. Under Aim 1, we will define the interactions of MgrA and NorG in expression of norA and other genes encoding efflux pumps. We specifically hypothesize that phosphorylation of MgrA alters its interactions with NorG and changes its effects on transcription of pump genes. The work will include measuring expression of norA by Northern hybridization in strains with constructed mutations in HprK kinase, and measurements of promoter binding of phosphorylated and unphosphorylated MgrA. Under Aim 2, we will identify the direct regulators of norB, tet38, and abcA by isolating proteins from cell extracts that bind to immobilized promoter DNA fragments, determining their identity by mass spectroscopy, and generating strains with mutations in and overexpression of the genes so identified. Such strains will be assessed for changes in expression of genes encoding pumps by Northern hybridization and transcriptional profiling. Under Aim 3, we will evaluate the global effects of NorG by transcriptional profiling of strains with mutations in and overexpression of norG. Under Aim 4, we will identify other candidate efflux pumps shown to be over-expressed in microarray analyses of regulatory networks and environmental stresses and define their roles in antibiotic resistance. Specific novel candidate pump genes distinct from those listed have been shown to be overexpressed in sarA, mgrA, and rot global regulatory mutants and under conditions evoking the stringent response or growth in a mouse subcutaneous abscess model. These candidate pump genes will be cloned and the effects of their overexpression on susceptibility to antibiotics and biocides will be assessed in order to add to the complement of known efflux resistance mechanisms. Under Aim 5, we will identify the environmental triggers of increased expression of norB, tet38, and other efflux pump genes identified under Aim 4. We have shown that norB and tet38 are overexpressed in S. aureus abscesses and that knockouts of these genes reduce fitness in the abscess milieu. Thus, we hypothesize that pumps are important for bacterial survival in an abscess in part due to relative protection from antimicrobial peptides, which are present in high concentrations. We will test specifically the role of low iron conditions and high concentrations of antimicrobial peptides as inducers of pump expression by measurement of pump gene RNA levels by Northern blotting, and we will determine the effects of pump overexpression of resistance to antimicrobial peptides in vitro. PUBLIC HEALTH RELEVANCE Antibiotic resistance in common bacterial pathogens such as Staphylococcus aureus is increasing in hospitals and the community. Efflux pumps serve to protect bacteria from adverse environments. Multidrug (MDR) and quinolone resistance conferred by multidrug resistance efflux pumps can also compromise drug utility and affect patient responses to antibiotics. Thus, defining the full complement of MDR efflux pumps in S. aureus and how they are regulated will provide both practical and fundamental information that are important for understanding of staphylococcal fitness to cause disease in diverse environments and for optimizing antibiotic action and use.