Transport function and dynamic assembly of a recombinant bacterial multidrug efflux pump, AcrAB-TolC

Abstract:

[Background] Multidrug efflux pump mostly presents in the form of membrane protein complex, which makes major contributions to bacterial drug resistance. The transport function and assembly of the efflux pump are among the important issues for bacterial drug resistance and drug development. [Objective] Taking AcrAB-TolC, an important member of resistance-nodulation cell division (RND) family, as the research example, this study aimed to investigate and analyze the transport activity and in vitro assembly of the multidrug efflux pump complex. [Methods] Basing on the gene sequence of AcrAB-TolC complex in Escherichia coli, the recombinant plasmids containing acrA, acrB and tolC genes were constructed. Each subunit of the complex was expressed and purified. This study then explored the transport functions of the complex and subunits, the interaction between subunits and substrates, as well as the interaction between subunits and their dynamic assembly by means of fluorescence spectroscopy and isothermal titration calorimetry (ITC). [Results] The expression and purification of the subunits of the AcrAB-TolC complex have been achieved with the homogeneity to over 98%. It was confirmed that living cells with expressed individual subunits had increased activity to transport ethidium bromide (EB). The activities of AcrB and TolC to transport EB were affected by N-hexanoyl-L-homoserine lactone (C6-HSL), a quorum-sensing signal molecule. ITC results further confirmed the interactions between C6-HSL and AcrB or TolC. In addition, there were obvious correlations in AcrA-AcrB and AcrA-TolC, while no significant interaction was found in AcrB-TolC. In the in vitro assembly experiments, the monomolecular fluorescence intensity of AcrAB-TolC subunit increased with time, which validated the dynamic assembly of the complex subunits on the membrane. [Conclusion] The AcrAB-TolC efflux pump and its subunits were expressed and purified. The activity of AcrAB-TolC to transport and its interaction with substrates were verified. The dynamic assembly of AcrAB-TolC was observed. These results facilitated the study of bacterial drug resistance and antimicrobial strategies in associating with multidrug efflux pumps.

Key words:bacterial resistance;resistance-nodulation-cell division family (RND);AcrAB-TolC;C6-HSL;isothermal titration calorimetry (ITC);fluorescence;dynamic assembly