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

描述（由申请人提供）： S.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，并且是最常见的引起严重感染的生物体之一。这种病原体具有多种机制，通过这些机制它抵抗杀生物剂和抗生素的杀伤作用，包括称为多药耐药性（MDR）赋予外排泵（EP）的膜基蛋白的过表达。事实上，外排是细菌如S.金黄色葡萄球菌可以同时逃避多种结构不同的抗微生物剂的作用。EP活性也使S.金黄色葡萄球菌通过将细胞内浓度降低至亚抑制水平来获得对某些泵底物的基于靶点的高水平抗性赋予突变。EP属于五个不同的蛋白质家族之一，这些蛋白质家族通过结构特征和用于底物运输的能量来源来区分。多药和毒性化合物挤出（MATE）家族是最近描述的，其成员不仅在细菌中发现，而且在包括植物、酵母和人类在内的真核生物中发现。MATE泵的典型基质包括一价和二价有机阳离子，如杀生物剂和消毒剂、氟喹诺酮和抗癌剂。收购MDR S。金黄色葡萄球菌菌株，包括MATE和其他MDR外排泵基因表达增加的菌株，可产生不希望的后果，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S.金黄色葡萄球菌，并且mepA的过表达发生在临床菌株中。编码MepR（mepA的MarR家族转录阻遏物）的mepR中的点突变经常是mepA过表达的基础，并且在临床菌株中发现并且容易在实验室中产生。然而，mepRA调节的其他机制也存在，因为已经鉴定了缺乏mepR突变的mepA过表达临床菌株。本申请提出了旨在增加我们对mepRA泵系统的理解的实验，特别是S.金黄色葡萄球菌。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，并使用诱变来表征MepR-诱导剂结合位点;（2）确定MepA泵的功能特征，并使用诱变来更好地理解底物/抑制剂与它的相互作用，这将为蛋白质的未来结构生物学分析提供信息;（3）表征MepR依赖性和非依赖性mepRA调节机制，包括天然存在的MepR取代和操纵基因位点突变以及反式作用因子。将采用MepR功能和操纵位点结合研究以及质粒文库分析来实现这一目标。MepA的详细研究，结合类似的早期工作与其他临床重要的S。金黄色葡萄球菌多药耐药EP（诺拉和QacA/B），将有助于广谱EP抑制剂的合理设计。 公共卫生相关性： S.金黄色葡萄球菌是主要的社区和医院获得性病原体。该项目将提供数据，增加我们对多药和有毒化合物挤出（MATE）家族外排泵的理解，这些知识也可能适用于真核MATE蛋白，并将为未来设计抑制多种S.金黄色葡萄球菌MDR泵同时进行。这将是抗菌化疗的一个进步，从而改善患者的结局，这与VA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.