Transport across two membranes by AcrAB-TolC

项目说明 以AcrAB-TolC为例的多药外排泵是临床的主要贡献者 细菌中的抗生素耐药性以及在发病和慢性感染过程中的各种适应性反应。 在之前的资助期间，我们在两个方面取得了重大进展：1)理解分子 外排泵组装机制及胞膜融合蛋白在多药外排中的作用 跨两层膜；和2)开发协同计算和经验方法来 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发挥作用的抑制剂，通过与AcrA相互作用并抑制AcrAB- TolC综合体。这些外排泵抑制剂增强了多种抗生素在各种细菌中的活性。这个 拟议研究的主要目标是建立新的外排泵抑制剂的分子机制。 并优化这些抑制剂，以便与特定的抗生素和针对特定的多药联合使用 耐药细菌。潜在的假设是，已发现的抑制剂的广谱增强活性是 这是由其独特的机制造成的，这种机制将外排泵困在一个组装不良且有泄漏的构型中。在 建议的方法、生化、结构和动力学实验将与先进的 外排泵抑制剂的机理表征和作用于外排泵的抑制剂优化计算 耐多药鲍曼不动杆菌泵。为了优化抑制剂，我们将把它应用于我们的 知识是可用的最全面的平台。该平台利用一组具有可变外排的菌株 容量和外膜通透性，并允许建立结构-活性关系 分别用于避免外流、抑制和透过外膜。成功完成 拟议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 抗生素耐药的多种泵和机制。

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.