Transport across two membranes by AcrAB-TolC

项目描述 以来自大肠杆菌的AcrAB-TolC为例的多药外排泵是临床应用的主要贡献者。 细菌的抗生素耐药性以及发病和慢性感染期间的各种适应性反应。 在上一个资助期间，我们在两个领域取得了重大进展：1）了解分子 外排泵组装机制及周质膜融合蛋白在多药外排中的作用 跨越两个膜;和2）协同计算和经验方法的发展， 发现具有新作用机制的外排泵抑制剂。我们成功地将这些进步应用于 发现以一种新的方式起作用的抑制剂，通过与AcrA相互作用并抑制AcrAB的组装， TolC复合体。这些外排泵抑制剂增强多种抗生素在各种细菌中的活性。的 本研究的主要目标是建立新型外排泵抑制剂的分子机制 并优化这些抑制剂以与特定抗生素组合使用并对抗特定的多药耐药性， 耐药细菌潜在的假设是，所发现的抑制剂的广泛增强活性是 这是由于其独特的机制，即在组装不良和泄漏的构象中捕获外排泵。在 建议的方法，生物化学，结构和动力学实验将协同使用先进的 计算以表征外排泵抑制剂的机制并优化作用于外排的抑制剂 多重耐药鲍曼不动杆菌的泵。为了优化抑制剂，我们将应用我们的 知识是最全面的平台。该平台利用一组具有可变外排率的菌株 能力和外膜渗透屏障，并允许建立结构-活性关系 分别用于外排避免、抑制和穿过外膜的渗透。成功完成 所提出的实验将有助于设计即使在以下情况下也有效的外排泵抑制剂： 多重泵和抗生素耐药性机制。

Project Description Multidrug efflux pumps as exemplified by AcrAB-TolC from Escherichia coli are the major contributors to clinical antibiotic resistance in bacteria and to various adaptive responses during pathogenesis and chronic infections. During the previous funding period, we made major advances in two areas: 1) understanding the molecular mechanism of efflux pump assembly and the role of periplasmic membrane fusion proteins in multidrug efflux across two membranes; and 2) the development of a synergistic computational and empirical approach to discovering efflux pump inhibitors with novel mechanisms of action. We successfully applied these advances to discover inhibitors that act in a novel way, by interacting with AcrA and inhibiting the assembly of the AcrAB- TolC complex. These efflux pump inhibitors potentiate activities of multiple antibiotics in various bacteria. The major goal of the proposed research is to establish the molecular mechanisms of the new efflux pump inhibitors and to optimize these inhibitors for use in combination with specific antibiotics and against specific multidrug resistant bacteria. The underlying hypothesis is that the broad potentiation activity of the discovered inhibitors is caused by their unique mechanism that traps efflux pumps in a poorly assembled and leaky conformation. In the proposed approach, biochemical, structural and kinetic experiments will be used synergistically with advanced computations to characterize the mechanism of efflux pump inhibitors and to optimize inhibitors acting on efflux pumps of multidrug resistant Acinetobacter baumannii. To optimize inhibitors, we will apply what is to our knowledge the most comprehensive platform available. The platform utilizes a set of strains with variable efflux capacities and outer membrane permeability barriers and allows to establish structure-activity relationships separately for efflux avoidance, inhibition and permeation across the outer membrane. Successful completion of the proposed experiments will help design efflux pump inhibitors that would be effective even in the context of multiple pumps and mechanisms of antibiotic resistance.