Transport across two membranes by AcrAB-TolC complex

AcrAB-TolC 复合物跨两膜转运

• 批准号: 7749979
• 负责人: HELEN I ZGURSKAYA
• 金额: $ 36.69万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7749979
• 关键词: Affinity; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Bacterial Physiology; Binding; Biochemical; Biological Assay; Cell division; Cells; Chemicals; Chimeric Proteins; Complex; Cysteine; Development; Drug Efflux; Escherichia coli; Goals; Gram-Negative Bacteria; Gram-Positive Bacteria; Hand; Human; In Vitro; Infection; Ion Channel; Kinetics; Link; Membrane; Membrane Fusion; Membrane Transport Proteins; Methods; Molecular Conformation; Multi-Drug Resistance; Outcome; Pathogenesis; Play; Positioning Attribute; Program Development; Property; Protein Family; Proteins; Public Health; Pump; Recruitment Activity; Research; Resistance; Role; Specificity; Substrate Specificity; Surface Plasmon Resonance; Techniques; Testing; Transport Reaction; Work; bacterial resistance; combat; comparative; drug discovery; efflux pump; in vivo; inhibitor/antagonist; insight; mutant; news; novel; novel strategies; pathogen; periplasm; protein function; prototype; public health relevance; reconstitution; tool

DESCRIPTION (provided by applicant): The main cause of antibiotic resistance of Gram-negative bacteria is active efflux of drugs from cells by multidrug efflux (MDR) transporters. MDR transporters from Resistance-Nodulation-cell Division (RND) superfamily possess an astonishing breadth of substrate specificity. The key mechanistic advantage of RND pumps is that they capture antibiotics in the periplasm and extrude them across the outer membrane of Gram-negative bacteria. This activity is possible due to the concerted action of the RND pumps and proteins belonging to the Membrane Fusion Protein (MFP) family. MFPs are absolutely required for multidrug resistance of Gram-negative pathogens. However, how MFPs enable drug efflux remains unclear. The long term goal is to understand the mechanism of drug efflux in Gram-negative bacteria. The objective of this application is to characterize the biochemical mechanism of MFPs. Our central hypothesis is that MFPs in Gram-negative bacteria play a dual role. On one hand, MFPs are functional subunits of transporters and are required to initiate transport cycles. On the other hand, these proteins are needed to create a physical link and coordinate actions between components of MDR complexes located in two different membranes. The approach used to test this hypothesis is to investigate the mechanistic properties of AcrA and compare them to MFPs functioning with multidrug efflux transporters belonging to different families of proteins. We will pursue three specific aims: (i) Investigate the mechanism of MFP-dependent transport reaction; (ii) Investigate the stability and specificity of interactions between MFPs and their cognate transporters; (iii) Investigate functional interactions of structurally diverse MFPs with the outer membrane. Under the first aim, we will characterize the kinetics and energetics of native and mutant efflux pumps using already proven transport in intact cells and in vitro reconstitution approaches. Under the second and third aims, surface plasmon resonance and in vivo cysteine accessibility approaches will be used to characterize functional interactions between MFPs and two other components of drug efflux complexes: the inner membrane transporters and the outer membrane channels. The expected outcome of the proposed studies is the mechanistic understanding how MFPs function in transport of substrates across two membrane envelope of Gram-negative bacteria. This contribution is significant because MFPs are absolutely required for antibiotic resistance and their function could be targeted in development of effective inhibitors of multidrug efflux transporters. PUBLIC HEALTH RELEVANCE: This application is focused on the most troubling form of antibiotic resistance in bacteria - multidrug resistance, which is caused by activities of efflux transporters. Multidrug efflux transporters are important targets in drug discovery and development programs. Understanding the biochemical mechanism of these transporters will greatly facilitate the development of new strategies to combat multidrug resistant bacteria.

描述（由申请人提供）：革兰氏阴性细菌的抗生素耐药性的主要原因是多种液体外排（MDR）转运蛋白从细胞中对药物的活跃外排。来自耐药性结构细胞分裂（RND）超家族的MDR转运蛋白具有惊人的底物特异性。 RND泵的关键机械优势是它们捕获了周质中的抗生素，并将其挤压在革兰氏阴性细菌的外膜上。由于属于膜融合蛋白（MFP）家族的RND泵和蛋白质的一致作用，因此可以使用这种活性。 MFP绝对需要革兰氏阴性病原体的多药耐药性。但是，MFP如何使药物外排还不清楚。长期目标是了解革兰氏阴性细菌中药物外排的机制。该应用的目的是表征MFP的生化机制。我们的中心假设是革兰氏阴性细菌中的MFP起着双重作用。一方面，MFP是转运蛋白的功能亚基，需要启动运输周期。另一方面，需要这些蛋白质来在两个不同膜中的MDR复合物的组件之间建立物理链接和坐标作用。用于检验该假设的方法是研究ACRA的机械性能，并将其与属于不同蛋白质家族的多药于外排运输蛋白的MFP进行比较。我们将追求三个具体目标：（i）研究MFP依赖性转运反应的机制； （ii）研究MFP及其同源转运蛋白之间相互作用的稳定性和特异性； （iii）研究结构上不同的MFP与外膜的功能相互作用。在第一个目标下，我们将使用已经在完整的细胞和体外重构方法中已经过证实的运输方式来表征天然和突变出外排泵的动力学和能量。在第二和第三目的下，表面等离子体的共振和体内半胱氨酸可及性方法将用于表征MFPS与其他两个药物外排复合物之间的功能相互作用：内膜内膜转运蛋白和外膜外膜通道。拟议研究的预期结果是理解MFP在跨两个革兰氏阴性细菌跨膜包膜底物运输中的作用。这种贡献很重要，因为MFP绝对需要抗生素耐药性，并且其功能可以针对开发有效的多药外排转运蛋白抑制剂。 公共卫生相关性：该应用集中在细菌中最令人不安的抗生素抗性形式 - 多药耐药性，这是由外排转运蛋白活动引起的。多药外排转运蛋白是药物发现和开发计划中的重要目标。了解这些转运蛋白的生化机制将极大地促进制定抗多药细菌的新策略。