Studies on the functional assembly of a non-functional VceAB-OprM multiple drug resistant efflux pump in Eschericha coli

Abstract

Active efflux is now recognized as one of the major mechanisms of multiple drug resistance for many bacteria. Multiple drug resistance (MDR) efflux pumps of Gramnegative bacteria are usually tripartite in nature, consisting of a cytoplasmic membrane translocase protein (CMT), periplasmic accessory or membrane fusion protein (MFP), and an outer membrane channel or efflux protein (OEP). Although the crystal structures of representative components of these pumps have been solved and the architecture of the assembled pump is beginning to be understood, the mechanism(s) by which these components are assembled into a functional pump are yet to be deciphered. In an attempt to examine the specificity of pump assembly, we have isolated and characterized gain-offunction (gof) mutations that allow a non-functional chimeric efflux pump (VceABOprM) to become functional. All gof mutations isolated mapped to the periplasmic accessory protein (in its hairpin domain). Cross-linkage studies indicated that only mutant gof VceA could be cross-linked to OprM, suggesting that this region of VceA plays an important role in the recruitment and assembly of a functional VceAB-OprM pump. We also found that the OprM used in the gof study is carrying an alterative C-terminal domain when compared to the wild type OprM (NCBI gi|3184189|). The altered Cterminus of OprM prevents it from interacting with VceA and thus from forming a functional pump with the VceAB translocase. We extended our study on the C-terminus of the wild type OprM, and analyzed the stability and functionality of C-terminal modified (truncations, deletions and C-terminal swaps) OprM in E.coli in the presence of VceAB (V.cholera), AcrAB (E.coli), MexAB (P.aeruginosa) and gof VceAB. We demonstrated that the 463LGGG466 motif, that is the last loop bridging the OEP helix 8 and the C-terminus, is required for the OprM stability and the functional assembly of the VceAB-OprM pump. We also observed that the mutant gof VceA could partially restore the stability and functionality of the C-terminal modified OprM. Therefore, the results suggest that the C-terminal modification caused OEP instability could be suppressed by forming a functional chimeric efflux pump in E.coli; and imply a possible physical interaction between the hairpin domain of MFP and OEP helix 8 (or 463LGGG466 motif).