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

点击翻译按钮获取中文摘要

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.