Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa.
Pseudomonas aeruginosa expresses a low level of the MexAB-OprM efflux pump and shows natural resistance to many structurally and functionally diverse antibiotics. The mutation that has been referred to previously as nfxC expresses an additional efflux pump, MexEF-OprN, exhibiting resistance to fluoroquinolones, imipenem, and chloramphenicol and hypersusceptibility to beta-lactam antibiotics. To address the antibiotic specificity of the MexEF-OprN efflux pump, we introduced a plasmid carrying the mexEF-oprN operon into P. aeruginosa lacking the mexAB-oprM operon. The transformants exhibited resistance to fluoroquinolones, trimethoprim, and chloramphenicol but, unlike most nfxC-type mutants, did not show beta-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEF-OprN pump subunit(s) responsible for substrate selectivity by expressing MexE, MexF, OprN, and MexEF in strains lacking MexA, MexB, OprM, and MexAB, respectively. The MexEF-OprM/DeltaMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to beta-lactam antibiotics compared with the host strain. The MexAB-OprN/DeltaOprM, MexAF-OprM/DeltaMexB, and MexEB-OprM/DeltaMexA mutants exhibited antibiotic susceptibility indistinguishable from that in the mutant lacking both types of efflux pumps. The results imply that the MexEF-OprM pump selects substrates by a MexEF functional unit. Interestingly, OprN did not link functionally with the MexAB complex, despite the fact that OprM interacted functionally with MexEF.
Maseda
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Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan.
Yoneyama
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H
Nakae
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D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics; D017353:Gene Deletion; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; C105708:OprN protein, Pseudomonas aeruginosa