Abstract
Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.
Highlights
Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance
Three-dimensional tomographic reconstructions revealed detailed structures of the Gram-negative bacterial envelope, with abundant channel-like densities spanning the cell envelope (Fig. 1 and Supplementary Movie 1). These densities are rarely observed in wild-type E. coli cells (Supplementary Fig. 2), implying that they correspond to AcrAB–TolC pumps
The distance between the outer membrane and the inner membrane stays constant at the sites where the AcrAB–TolC pumps occur, suggesting that the periplasm may be pinched by these assemblies
Summary
Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. We report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryotomography and subtomogram averaging. The AcrAB–TolC efflux pump transports diverse compounds, conferring resistance to a broad spectrum of antibiotics[6] Structural studies of this pump have been limited to individual components by X-ray crystallography[7,8,9,10,11,12,13,14] or fully assembled pumps by cryo-electron microscopy (cryo-EM) singleparticle analysis[15,16,17,18,19]. Our results reveal in situ structures of the fully assembled pump and its intermediate assembly state and suggest an assembly mechanism for tripartite efflux pumps in Gram-negative bacteria
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