Quinolone and multidrug resistance in Staphylococcus aureus

描述（由申请人提供）：我们拟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抗菌药物耐药性中的作用，重点是喹诺酮类耐药性。在先前的工作中，我们鉴定了由诺拉、B和C泵的表达增加引起的喹诺酮耐药性，由Tet 38泵引起的四环素耐药性，由AbcA泵引起的β-内酰胺耐药性，以及其他人已经鉴定了由MepA泵引起的杀生物剂耐药性。我们还确定了泵表达的直接转录调节因子，MgrA和NorG，它们相互作用并具有广泛的影响。有五个具体目标。在目标1下，我们将定义MgrA和NorG在诺拉和编码外排泵的其他基因的表达中的相互作用。我们特别假设，磷酸化MgrA改变其与NorG的相互作用，并改变其对泵基因转录的影响。这项工作将包括通过北方杂交在HprK激酶中构建突变的菌株中测量诺拉的表达，以及测量磷酸化和非磷酸化MgrA的启动子结合。根据目标2，我们将确定norB，tet 38，和abcA的直接调节剂，通过从细胞提取物中分离蛋白质，结合到固定的启动子DNA片段，确定其身份的质谱，并产生菌株的突变和过表达的基因，因此确定。将通过北方杂交和转录谱分析评估这些菌株的泵编码基因表达的变化。在目标3下，我们将通过对norG突变和过表达菌株的转录谱分析来评估norG的整体效应。在目标4下，我们将确定其他候选外排泵，这些泵在调控网络和环境压力的微阵列分析中过度表达，并定义它们在抗生素耐药性中的作用。与所列基因不同的特定新候选泵基因已被证明在sarA、mgrA和rot全局调节突变体中过表达，并且在引起小鼠皮下脓肿模型中的严格反应或生长的条件下过表达。将克隆这些候选泵基因，并评估其过表达对抗生素和杀生物剂敏感性的影响，以补充已知的外排耐药机制。在目标5下，我们将确定目标4下确定的norB，tet 38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tet 38在S.金黄色葡萄球菌脓肿和这些基因的敲除降低了在脓肿环境中的适应性。因此，我们假设泵对脓肿中细菌的存活很重要，部分原因是由于抗微生物肽的相对保护，这些肽以高浓度存在。我们将通过北方印迹法测量泵基因RNA水平，专门测试低铁条件和高浓度抗菌肽作为泵表达诱导剂的作用，并确定泵过表达对体外抗菌肽耐药性的影响。公共卫生相关性在医院和社区，常见细菌病原体（如金黄色葡萄球菌）的抗生素耐药性正在增加。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）和喹诺酮耐药也会损害药物效用并影响患者对抗生素的反应。因此，定义完整的MDR外排泵在S。金黄色葡萄球菌和它们是如何被调节的将提供实用和基本的信息，这对于理解葡萄球菌在不同环境中引起疾病的适应性以及优化抗生素作用和使用是重要的。

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.