Understanding multidrug efflux inhibition in gram negative b acteria

本研究的目的是了解旨在抑制抗生素从细菌外排的化合物的外排抑制机制。少数抗菌剂对革兰氏阴性菌有效。这是由于细胞壁的结构具有不渗透性和多药耐药（MDR）三方外排泵的存在，它们泵出许多抗生素，从而产生内在的多药耐药。规避外排的一种方法是将许可的抗生素与外排抑制剂结合使用，以“挽救”原本有效的药物并延长其使用寿命。文献中描述的许多外排泵抑制剂的作用机制尚不清楚。因此，了解这些化合物如何起作用将有助于发现新的抗耐多药细菌的ei -抗生素组合。里程碑1（高18米）。化合物EI活性的体外验证：耐多药外排泵具有广泛的底物范围，因此（EI）化合物抑制抗生素出口的能力将直接在耐多药细菌（如大肠杆菌、鲍曼杆菌和铜绿假单胞菌）中进行测量。这将使用最新的全细胞方法来检测细菌细胞中积累和流出的抗生素，包括荧光分光光度法和质谱法。里程碑2（高36米）。体内EI效应的研究：在EI存在的情况下，外排敏感抗生素比单独使用更有效。这不仅是因为细胞内残留的抗生素浓度较高，而且还因为外排泵蛋白的失活或功能丧失降低了细菌感染宿主并形成生物膜的能力。Evotec建立的行业标准细菌感染模型（包括大腿和肺负荷小鼠模型）和全动物成像将用于确定EI化合物+/-抗生素的效果。细菌粘附和侵入感染组织培养模型的能力以及形成和根除生物膜的能力也将被测量。里程碑3（高48米）。EI机制的阐明：我们对底物通过外排泵输运的机制的理解仍然不完整。目前的模型是，底物通过与两个结合袋中的一个结合，通过泵的转运体组件（例如AcrB）移动。因此，我们认为EI要么通过竞争性运输发生，要么通过阻塞绑定袋发生。EI的表型也可以通过转运体成分的减少而获得。因此，EI的机制将通过(i)测量已知RND外排泵抑制剂存在时的外排活性，以及在底物结合袋内具有明确突变的突变体，以及（ii）转录组学和蛋白质组学来确定外排泵成分的水平来研究。该项目将使用分子和细胞微生物学、临床微生物学、体内研究和生物化学。

The aim of this studentship is to understand the mechanism of efflux inhibition (EI) of compounds purported to inhibit efflux of antibiotics from bacteria. A minority of antibacterials are active against Gram-negative bacteria. This is due to the structure of the cell wall conferring impermeability and the presence of multi-drug resistance (MDR) tripartite efflux pumps, which pump out many antibiotics so conferring intrinsic MDR. An approach to circumvent efflux is to combine a licenced antibiotic with an efflux inhibitor to 'rescue' an otherwise effective drug and extend its useful life. Little is known about the mechanism of action of many compounds described in the literature as efflux pump inhibitors. Therefore, understanding how these compounds work will inform discovery of new EI-antibiotic combinations active against MDR bacteria. Milestone 1 (by 18m). Validation of EI activity of compounds in vitro: MDR efflux pumps have a wide substrate range and so the ability of (EI) compounds to inhibit the export of antibiotics will be measured directly in MDR bacteria such as E. coli, A. baumannii and P. aeruginosa. This will use the latest whole cell methodologies to detect antibiotic accumulated and effluxed from the bacterial cell including fluorescence spectrophotometry and mass spectrometry.Milestone 2 (by 36m). Investigation of the effect of EI in vivo: In the presence of an EI an efflux-susceptible antibiotic will be more effective than alone. This is not just due to higher concentrations of antibiotic remaining inside the cell, but also because inactivation or loss of function of efflux pump proteins reduces the ability of bacteria to infect the host and form a biofilm. Industry standard models of bacterial infection (including thigh and lung burden murine models) and whole animal imaging established at Evotec will be used to determine the effect EI compounds +/- antibiotic. The ability of bacteria to adhere to, and invade tissue culture models of infection and to form and eradicate a biofilm will also be measured. Milestone 3 (by 48m). Elucidation of the mechanism of EI: Our understanding of the mechanism of transport of substrates via efflux pumps is still incomplete. The current model is that substrates are moved through the transporter component of the pump (e.g. AcrB) via binding to one of two binding pockets. Therefore, EI is proposed to occur either through competitive transport or blocking of a binding pocket. The phenotype of EI can also be conferred by a reduction in amount of the transporter component. Therefore, the mechanism of EI will be investigated via (i) measurement of efflux activity in the presence of known inhibitors of RND efflux pumps and for mutants with defined mutations within the substrate binding pockets, and (ii) transcriptomics and proteomics to determine the level of efflux pump components. This project will use molecular and cellular microbiology, clinical microbiology, in vivo studies and biochemistry.