Assignment of the Substrate-Selective Subunits of the MexEF-OprN Multidrug Efflux Pump of Pseudomonas aeruginosa

Abstract

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 ␤-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 ␤-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/⌬MexAB transformants exhibited MexEF-OprN-type pump function that rendered the strains resistant to fluoroquinolones and chloramphenicol but did not change susceptibility to ␤-lactam antibiotics compared with the host strain. The MexAB-OprN/⌬OprM, MexAF-OprM/⌬MexB, and MexEB-OprM/⌬MexA 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.

Bacterial infection imaging with [ 18 F]fluoropropyl-trimethoprim

“…2B). The prime mechanism of P. aeruginosa resistance to TMP is due to small-molecule export pump (11,27). Small-molecule efflux is most consistent with a lower overall level of uptake at saturation; however, we cannot exclude that an altered K i for Pseudomonas dhfr is also involved and could contribute to the delayed uptake.…”

Molecular Cloning and Characterization of All RND‐Type Efflux Transporters in Vibrio cholerae Non‐O1

“…We cannot exclude the possibility that other outer membrane component(s) among the four remaining candidates is (are) important for function of the RND-type efflux transporters in V. cholerae. It has been reported in P. aeruginosa that several outer membrane components are present, and certain RND-type efflux transporters can utilize multiple outer membrane components (16,23,27,40). In order to understand the mechanism of drug transport via the RND-type efflux transporters, investigation of the interaction between the inner membrane component of the RND-type efflux transporter and the outer membrane component would be important.…”

High‐Level Fluoroquinolone Resistance in Pseudomonas aeruginosa Due to Interplay of the MexAB‐OprM Efflux Pump and the DNA Gyrase Mutation

“…All other strains were constructed in this study. Plasmids used were p∆OprM (pNOT19 derivative with oprM::tet, ABPC r , TET r and CP r , (11)), pMMB67EH (broad host range vector, ABPC r , IncQ, (11) and pMEXAB-OPRM1 (pMMB67EH derivative carrying mexA, mexB and oprM, (5)). Cells were grown aerobically at 37 C in L-broth containing 10 g of tryptone, 5 g of yeast-extract, and 5 g of NaCl per liter at pH 7.2.…”