Few conserved amino acids in the small multidrug resistance transporter EmrE influence drug polyselectivity. [PublishAheadOfPrint]

EmrE is the archetypical member of the small multidrug resistance transporter family and confers resistance to a wide range of disinfectants and dyes known as quaternary cation compounds (QCCs). The aim of this study was to examine which conserved amino acids play an important role in substrate selectivity. Based on previous analysis of EmrE homologs, a total of 33 conserved residues were targeted for cysteine or alanine replacements within E. coli EmrE. The antimicrobial resistance of each EmrE variant expressed in E. coli strain JW0451 (lacking dominant pump acrB) was tested against a collection of 16 different QCCs using agar spot dilution plating to determine MIC values. The results determined that few conserved residues were drug polyselective: the active site residue E14 (E14D and E14A) and 4 additional conserved residues (A10C, F44C, L47C, W63A) based on ≥4 fold decreases in MIC values. EmrE variants (I11C, V15C, P32C, I62C, L93C, S105C) enhanced resistance to polyaromatic QCCs, while the remaining EmrE variants reduced resistance to one or more QCCs with shared chemical features: acylation, tri-and tetra-phenylation, aromaticity, and dicationic charge. Mapping EmrE variants onto transmembrane helical wheel projections using the highest resolved EmrE structure suggests that polyselective EmrE variants were located closest to helical faces surrounding the predicted drug binding pocket, while EmrE variants with greater drug specificity mapped onto distal helical faces. This study reveals that few conserved residues are essential for drug polyselectivity and indicates that aromatic QCC selection involves a greater portion of conserved residues compared to other QCCs.

Importance EmrE is an archetypical member of the small multidrug resistance efflux pump family and its members are frequently identified from mobile genetic elements and multidrug resistant plasmids. This study examines conserved residues in EmrE and their MIC to various quaternary ammonium compounds which may shed light on the importance of conserved residues in other EmrE orthologues. In this study, few conserved EmrE residue replacements are required for drug polyselectivity but indicate that many conserved residues that may be important for polyaromatic QCC selection which may be useful for future efflux pump inhibition and inactivation studies.

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