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 β-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 β-lactam hypersusceptibility. The transformants exhibited additional resistance to tetracycline. In the next experiment, we analyzed the MexEFOprN 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/ΔMexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to β-lactam antibiotics compared with the host strain. The MexAB-OprN/ΔOprM, MexAF-OprM/ΔMexB, and MexEB-OprM/ΔMexA 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.