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

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，能够引起严重的、危及生命的感染，也是最常见的生物之一。这种病原体有多种机制来抵抗杀生剂和抗生素的杀灭作用，包括过度表达称为多药耐药(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同时逃避多种结构不同抗菌剂作用的最重要机制。EP活性还通过将细胞内浓度降低到亚抑制水平，使金黄色葡萄球菌获得针对某些泵底物的高水平抗药性突变。EPS属于五个不同的蛋白质家族之一，这些蛋白质家族根据结构特征和底物运输所用的能量来源而不同。多药和有毒化合物挤压(Mate)家族是最新描述的家族，其成员不仅存在于细菌中，而且存在于真核生物中，包括植物、酵母和人类。配对泵的典型底物包括一价和二价有机阳离子，如杀菌剂和消毒剂、氟喹诺酮类药物和抗癌剂。获得多药耐药金黄色葡萄球菌菌株，包括那些Mate和其他多药耐药外排泵基因表达增加的菌株，可能会产生不良后果，如延长住院时间，增加医疗费用，最重要的是增加发病率和死亡率。MEPA是在金黄色葡萄球菌中发现的第一种也是唯一一种耐甲氧西林金黄色葡萄球菌的耐甲氧西林金黄色葡萄球菌，在临床菌株中存在过表达。MepR编码MepR，MepR是MEPA的Marr家族转录抑制因子，它的点突变经常使蛋白失活，是MEPA过表达的基础，在临床菌株中发现，在实验室很容易产生。然而，随着缺乏mepR突变的过表达MEPA的临床菌株被发现，Mepra调节的其他机制也存在。这项申请提出的实验旨在增加我们对MEPRA泵系统的了解，特别是对金黄色葡萄球菌的MDR EPs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-诱导剂相互作用的细节，并利用突变鉴定MepR-诱导子结合位点(S)；(2)确定MEPA泵的功能特征并利用突变更好地了解底物/抑制物与它的相互作用，这将为未来的蛋白质结构生物学分析提供信息；(3)表征MepR依赖和独立的Mepra调控机制，包括自然发生的MepR替换和操作点突变以及反式作用因子。为实现这一目标，将利用MepR的功能和操纵子位点结合研究以及对质粒库的分析。对MEPA的详细研究，结合其他临床上重要的金黄色葡萄球菌MDR EPs(NorA和QacA/B)的类似早期工作，将有助于合理设计广谱EP抑制剂。 公共卫生相关性： 金黄色葡萄球菌是一种主要的社区获得性病原体。该项目将提供增加我们对多药和有毒化合物挤出(Mate)家族外排泵的了解的数据，这些知识也可能适用于真核细胞的Mate蛋白质，并将为未来设计同时抑制多个金黄色葡萄球菌MDR泵的化合物提供指导。这将是抗菌化疗的进步，从而改善患者的预后，这与退伍军人管理局的使命直接相关。

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.