Random mutagenesis of the multidrug transporter AcrB from Escherichia coli for identification of putative target residues of efflux pump inhibitors.

Autor:	Schuster S; Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, and Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany sabine.schuster@uniklinik-freiburg.de., Kohler S; Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, and Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany Department of Biology, Eberhard Karls-University, Tübingen, Germany., Buck A; Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, and Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany., Dambacher C; Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, and Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany., König A; Institute for Environmental Health Sciences, Albert-Ludwigs-University, Freiburg, Germany., Bohnert JA; Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, and Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany Institute of Medical Microbiology, Jena University Hospital, Jena, Gemany., Kern WV; Center for Infectious Diseases and Travel Medicine, University Hospital Freiburg, and Department of Medicine, Albert-Ludwigs-University, Freiburg, Germany.
Jazyk:	angličtina
Zdroj:	Antimicrobial agents and chemotherapy [Antimicrob Agents Chemother] 2014 Nov; Vol. 58 (11), pp. 6870-8. Date of Electronic Publication: 2014 Sep 02.
DOI:	10.1128/AAC.03775-14
Abstrakt:	Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
Databáze:	MEDLINE
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