Dynamics of the AcrA-AcrB-TolC, a multi-drug efflux system of escherichia coli

大肠杆菌多药物外排系统 AcrA-AcrB-TolC 的动力学

• 批准号: 327141-2006
• 负责人: Liu, Jun
• 金额: $ 2.77万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2008
• 资助国家: 加拿大
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=397602
• 关键词: Dynamics; AcrA; AcrB; TolC; multi

The AcrA-AcrB-TolC efflux system of Escherichia coli is largely responsible for the intrinsic resistance of E. coli to most lipophilic antibiotics, detergents, and dyes. AcrB is the efflux transporter located at the inner membrane (IM) and AcrA is a periplasmic protein of the membrane fusion protein (MFP) family, while TolC is an outer membrane (OM) channel.  This tripartite complex couples the energy (proton-motive-force [PMF]) at the IM with the efflux of substrates, and allows the system to pump out drugs directly into the medium, making it much more efficient in producing resistance. Recently, crystal structures AcrB and TolC, together with that of an AcrA homolog, MexA, have provided remarkable insights for the assembly of the efflux complex. However, questions remain as to how these three proteins assemble to form a functional pump and how channel openings and substrate movements occur. Specifically, the role of AcrA is not completely understood and how the energy PMF is utilized to drive the efflux process of substrates remains unknown. Knowledge on the dynamics of the system during the efflux process will help to address these questions. In this proposal, we plan to utilize a sensitive biophysical technique, the site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy, to study the dynamics of the AcrA-AcrB-TolC system. Using this technique, we have recently demonstrated that AcrA undergoes pH-induced conformational changes, suggesting that AcrA may act as a dynamic molecular switch during the efflux process, rather than a static adaptor. In the proposed research, our short-term goal is to further investigate the dynamics of AcrA and its functional relevance in vivo, so that its role in the efflux system can be better understood.  The long-term goal is to utilize the SDSL technique to investigate the dynamics of all three components (AcrA, AcrB and TolC) during the efflux process in order to fully understand the mechanism of action of multidrug efflux pumps in Gram-negative bacteria. Aside from being intellectually rewarding, unraveling the mechanistic details will greatly facilitate the fight to combat antibiotic resistance.

大肠杆菌的 AcrA-AcrB-TolC 外排系统在很大程度上导致了大肠杆菌对大多数亲脂性抗生素、清洁剂和染料的内在抗性。 AcrB 是位于内膜 (IM) 的外排转运蛋白，AcrA 是膜融合蛋白 (MFP) 家族的周质蛋白，而 TolC 是外膜 (OM) 通道。  这种三方复合物将 IM 处的能量（质子动力 [PMF]）与底物的流出耦合起来，并允许系统将药物直接泵入介质中，从而更有效地产生耐药性。最近，晶体结构 AcrB 和 TolC 以及 AcrA 同源物 MexA 的晶体结构为外排复合物的组装提供了非凡的见解。然而，关于这三种蛋白质如何组装形成功能泵以及通道开口和底物运动如何发生的问题仍然存在。具体而言，AcrA 的作用尚未完全了解，并且如何利用能量 PMF 来驱动底物的外排过程仍然未知。有关外排过程中系统动力学的知识将有助于解决这些问题。在本提案中，我们计划利用敏感的生物物理技术，即定点自旋标记（SDSL）EPR（电子顺磁共振）光谱来研究 AcrA-AcrB-TolC 系统的动力学。使用这种技术，我们最近证明 AcrA 会经历 pH 诱导的构象变化，这表明 AcrA 在外排过程中可能充当动态分子开关，而不是静态适配器。在本研究中，我们的短期目标是进一步研究 AcrA 的动力学及其体内功能相关性，以便更好地理解它在外排系统中的作用。  长期目标是利用SDSL技术研究外排过程中所有三个组分（AcrA、AcrB和TolC）的动态，以充分了解多药外排泵在革兰氏阴性菌中的作用机制。除了智力上的回报之外，揭开机制细节将极大地促进对抗抗生素耐药性的斗争。