AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

Abstract

The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCEThe structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrABTolC-mediated multidrug export.

Genome sequencing and assessment of plant growth-promoting properties of a Serratia marcescens strain isolated from vermicompost

“…As expected, we found a number of antibiotic resistance genes in the S. marcescens core genome, including genes encoding a number of important efflux pumps (e.g. MdtH , MsrB and AcrA-AcrB-TolC multidrug efflux complex) [ 50 ]. Importantly, S. marcescens UENF-22GI lacks strain-specific antibiotic resistance genes, as well as a number of antibiotic resistance genes that are more prevalent in clinical than in non-clinical strains, such as, emrE , mexG , mexQ , mexP , golS , qnrB31 , qnrB37 , aadA16 , fosA2 , fosA3 , opmE , bla OXA -9 , bla TEM -1 , and sul1 [ 51 , 52 ].…”

AcrB: a mean, keen, drug efflux machine

“…Nevertheless, some degradation or transcriptional/translational separation of the AcrA-AcrB fusion must have occurred, since "free" AcrB was observed in the same samples. 200…”

Mutations in the TolC Periplasmic Domain Affect Substrate Specificity of the AcrAB-TolC Pump

“…The second model is derived predominantly from cryo-EM structural data suggesting only a limited interface between the AcrA hairpins and the tips of the TolC coiled-coils hence it is called the “tip-to-tip” model of interaction ( Xu et al, 2011 ), and results in an extended uniform tube ( Du et al, 2014 ; Kim et al, 2015 ; Daury et al, 2016 ; Shi et al, 2019 ; Figure 1 ). While most of the recent cryo-EM ( Kim et al, 2015 ; Daury et al, 2016 ; Wang et al, 2017 ; Tsutsumi et al, 2019 ) and cryo-tomography structures ( Shi et al, 2019 ) have favored the tip-to-tip model, there is still some ambiguity whether deeper interpenetration may take place transiently during part of the efflux-cycle ( Hayashi et al, 2016 ).…”