MepRA, A Substrate-Responsive Repressor-MATE MDR Efflux Pump Tandem in S. aureus

MepRA，金黄色葡萄球菌中的底物响应阻遏蛋白 - MATE MDR 外排泵串联

• 批准号: 8198378
• 负责人: GLENN WILLIAM KAATZ
• 金额: --
• 依托单位: JOHN D DINGELL VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2014-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8198378
• 关键词: Affect; Aminoglycosides; Anti-Bacterial Agents; Antibiotics; Antineoplastic Agents; Area; Bacillus subtilis; Bacteria; Bacterial Infections; Binding; Binding Sites; Biocide; Blood Circulation; Cations; Characteristics; Charge; Chloramphenicol; Clinical; Collection; Communities; Complex; DNA; Data; Disinfectants; Dyes; Energy-Generating Resources; Environment; Eukaryota; Europe; Exhibits; Family; Fluoroquinolones; Future; Gene Mutation; Genes; Genetic; Genetic Transcription; Goals; Health Care Costs; High Prevalence; Human; Infection; Investigation; Japan; Knowledge; Laboratories; Length of Stay; Libraries; Life; Local Anti-Infective Agents; Locales; Maintenance; Membrane; Methicillin; Mission; Mono-S; Morbidity - disease rate; Multi-Drug Resistance; Mutagenesis; Mutation; Nature; Nutrient; Organism; Outcome; Oxazolidinones; Patients; Pharmaceutical Preparations; Plants; Plasmids; Point Mutation; Poison; Process; Protein Analysis; Protein Family; Proteins; Protons; Pseudomonas aeruginosa; Publishing; Pump; Regulation; Reporting; Resistance; Site; Staphylococcus aureus; Streptococcus pneumoniae; Structural Genes; Structure; Substrate Interaction; System; Testing; Tetracyclines; Therapeutic; Topoisomerase; Trans-Activators; Transcription Repressor/Corepressor; United States; Work; Yeasts; antimicrobial; antimicrobial drug; base; beta-Lactams; chemotherapy; clinically relevant; design; efflux pump; fluoroquinolone resistance; inhibitor/antagonist; insight; killings; member; mortality; mutant; novel; overexpression; pH gradient; pathogen; prevent; public health relevance; research study; resistance mechanism; resistant strain; structural biology; transmission process

DESCRIPTION (provided by applicant): S. aureus is an important human pathogen capable of causing serious, life-threatening infections and is one of the most common organisms to do so. This pathogen possesses multiple mechanisms by which it resists the killing effects of biocides and antibiotics, including overexpression of membrane-based proteins called multidrug resistance (MDR)-conferring efflux pumps (EPs). In fact, efflux is the single most important mechanism by which bacteria such as S. aureus can evade the effects of multiple structurally different antimicrobial agents simultaneously. EP activity also predisposes S. aureus to acquire target-based high level resistance-conferring mutations to some pump substrates by reducing intracellular concentrations to subinhibitory levels. EPs belong to one of five different protein families that are differentiated by structural characteristics and energy source used for substrate transport. The Multidrug and Toxic compound Extrusion (MATE) family is the most recently described and members are found not only in bacteria but also in eukaryotes including plants, yeast, and humans. Typical substrates for MATE pumps include mono- and bivalent organic cations such as biocides and disinfectants, fluoroquinolones, and anticancer agents. Acquisition of MDR S. aureus strains, including those with increased expression of MATE and other MDR efflux pump genes, can produce undesirable consequences such as prolonged hospital stays, increased healthcare costs, and most importantly increased morbidity and mortality. MepA is the first and only MATE MDR EP identified in S. aureus, and overexpression of mepA occurs in clinical strains. Point mutations in mepR, which encodes MepR, a MarR-family transcriptional repressor of mepA, that inactivate the protein frequently are the bases of mepA overexpression and are found in clinical strains and easily produced in the laboratory. However, other mechanisms of mepRA regulation also exist as mepA-overexpressing clinical strains lacking mepR mutations have been identified. This application proposes experiments designed to increase our understanding of the mepRA pump system in particular and MDR EPs of S. aureus in general. Our goals are to (1) Determine the details of MepR-DNA and MepR-inducer interactions by structural biology investigations and characterize the MepR-inducer binding site(s) using mutagenesis; (2) Determine the functional characteristics of the MepA pump and employ mutagenesis to better understand substrate/inhibitor interactions with it, which will inform the future structural biology analysis of the protein; (3) Characterize MepR-dependent and - independent mepRA regulatory mechanisms, including naturally-occurring MepR substitution and operator site mutations and trans-acting factors. MepR functional and operator site binding studies and analyses of plasmid libraries will be employed to accomplish this goal. The detailed study of MepA, combined with similar earlier work with other clinically important S. aureus MDR EPs (NorA and QacA/B), will help in the rational design of broad-spectrum EP inhibitors. PUBLIC HEALTH RELEVANCE: S. aureus is a major community- and nosocomially-acquired pathogen. This project will provide data increasing our understanding of Multidrug and Toxic compound Extrusion (MATE) family efflux pumps, knowledge that also may be applicable to eukaryotic MATE proteins, and will inform future work toward the design of compounds that inhibit multiple S. aureus MDR pumps simultaneously. This will be an advance in antibacterial chemotherapy resulting in an improvement in patient outcomes, which is directly relevant to the mission of the VA.

描述（由申请人提供）： 金黄色葡萄球菌是一种重要的人类病原体，能够引起严重、危及生命的感染，也是最常见的微生物之一。这种病原体拥有多种机制来抵抗杀菌剂和抗生素的杀伤作用，包括过度表达称为多药耐药性（MDR）的外排泵（EP）的膜蛋白。事实上，外排是金黄色葡萄球菌等细菌可以同时逃避多种结构不同的抗菌剂作用的最重要的机制。 EP 活性还通过将细胞内浓度降低至亚抑制水平，使金黄色葡萄球菌易于获得针对某些泵底物的基于靶点的高水平抗性突变。 EP 属于五个不同蛋白质家族之一，这些蛋白质家族因结构特征和用于底物运输的能源而有所不同。多药和有毒化合物挤出 (MATE) 家族是最近被描述的，其成员不仅存在于细菌中，而且还存在于真核生物中，包括植物、酵母和人类。 MATE 泵的典型底物包括单价和二价有机阳离子，例如杀菌剂和消毒剂、氟喹诺酮类药物和抗癌剂。获得耐多药金黄色葡萄球菌菌株，包括 MATE 和其他耐多药外排泵基因表达增加的菌株，可能会产生不良后果，例如住院时间延长、医疗费用增加，最重要的是发病率和死亡率增加。 MepA 是在金黄色葡萄球菌中发现的第一个也是唯一一个 MATE MDR EP，并且 mepA 的过度表达发生在临床菌株中。 mepR 中的点突变编码 MepR（mepA 的 MarR 家族转录抑制因子），经常使该蛋白失活，是 mepA 过度表达的基础，在临床菌株中发现，并且很容易在实验室中产生。然而，mepRA 调节的其他机制也存在，因为已鉴定出缺乏 mepR 突变的 mepA 过表达临床菌株。本申请提出的实验旨在增加我们对 mepRA 泵系统（特别是 mepRA 泵系统）和金黄色葡萄球菌 MDR EP 的了解。我们的目标是 (1) 通过结构生物学研究确定 MepR-DNA 和 MepR-诱导子相互作用的细节，并使用诱变表征 MepR-诱导子结合位点； （2）确定MepA泵的功能特征，并利用诱变更好地了解底物/抑制剂与其相互作用，这将为未来的蛋白质结构生物学分析提供信息； (3) 表征 MepR 依赖性和独立性 mepRA 调节机制，包括自然发生的 MepR 取代和操纵位点突变以及反式作用因子。 MepR 功能和操纵位点结合研究以及质粒文库分析将用于实现这一目标。对 MepA 的详细研究，结合早期对其他临床重要的金黄色葡萄球菌 MDR EP（NorA 和 QacA/B）的类似研究，将有助于广谱 EP 抑制剂的合理设计。 公共卫生相关性： 金黄色葡萄球菌是一种主要的社区和医院获得性病原体。该项目将提供数据，加深我们对多药和有毒化合物挤出 (MATE) 系列外排泵的理解，这些知识也可能适用于真核 MATE 蛋白，并将为未来设计同时抑制多个金黄色葡萄球菌 MDR 泵的化合物的工作提供信息。这将是抗菌化疗的进步，从而改善患者的治疗效果，这与 VA 的使命直接相关。