Characterization of the MexC-MexD-OprJ multidrug efflux system in DeltamexA-mexB-oprM mutants of Pseudomonas aeruginosa

Abstract

Expression of the multidrug efflux system MexC-MexD-OprJ in nfxB mutants of Pseudomonas aeruginosa contributes to resistance to fluoroquinolones and the "fourth-generation" cephems (cefpirome and cefozopran), but not to most beta-lactams, including the ordinary cephems (ceftazidime and cefoperazone). nfxB mutants also express a second multidrug efflux system, MexA-MexB-OprM, due to incomplete transcriptional repression of this operon by the mexR gene product. To characterize the contribution of the MexC-MexD-OprJ system to drug resistance in P. aeruginosa, a site-specific deletion method was employed to remove the mexA-mexB-oprM region from the chromosome of wild-type and nfxB strains of P. aeruginosa. Characterization of mutants lacking the mexA-mexB-oprM region clearly indicated that the MexC-MexD-OprJ efflux system is involved in resistance to the ordinary cephems as well as fluoroquinolones and the fourth-generation cephems but not to carbenicillin and aztreonam. Rabbit polyclonal antisera and murine monoclonal antibody against the components of the MexA-MexB-OprM system were prepared and used to demonstrate the reduced production of this efflux system in the nfxB mutants. Consistent with this, transcription of the mexA-mexB-oprM operon decreased in an nfxB mutant. This reduction appears to explain the hypersusceptibility of the nfxB mutant to beta-lactams, including ordinary cephems.

FIG. 1

Schematic models of the TnpR-mediated site-specific deletion based on an improvement of the deletion system developed by Tsuda (34). Specifically, the plasmid carrying an additional res-Ω cassette and a res-tnpR block (pKMM157) was integrated into the oprM gene of the chromosome containing another res-Ω cassette in the mexR gene. Deletion between the two copies of the res site led to removal of the intervening chromosomal region and the integrated plasmid sequence, leaving one copy of the res site. Abbreviations for restriction endonuclease sites: P, PvuII; S, SacI; X, XhoI; H, HindIII; and N, NotI.

FIG. 2

Amplification of chromosomal fragments containing deletion endpoints. Approximately 1-kb fragments containing the remaining res site were amplified by PCR using primer sets that annealed at the 3′ ends of mexR and oprM (primers mexR2 and 1680, respectively). These fragments were digested with SacI and electrophoresed on a 1% (wt/vol) agarose gel. Lanes: 1 and 2, KG2239; 3 and 4, KG2240; 5 and 6, KG2259. Lanes 1, 3, and 5 show the ca. 1-kb amplified fragments, and lanes 2, 4, and 6 show the SacI-digested PCR products. Boxes labeled mexR-3′ and oprM-3′ refer to the 3′ ends of each gene. The sizes of the fragments are indicated in kilobases at the right of the figure.

FIG. 3

Detection of MexA-MexB-OprM and MexC-MexD-OprJ component proteins with antisera directed against synthetic oligopeptides containing part of the amino acid sequences of MexA, MexB, MexC, or MexD or monoclonal antibodies specific to OprM or OprJ. Each lane contains 30 μg of cell envelope protein, as determined by the method of Lowry et al. (15). Lanes: 1, PAO1; 2, KG2212 (PAO1 mexR::res-Ω); 3, KG2239 (PAO1 ΔmexR-mexA-mexB-oprM); 4, KG3052 (PAO1 type-A nfxB); 5, KG2217 (KG3052 mexR::res-Ω); 6, KG2240 (KG3052 ΔmexR-mexA-mexB-oprM); 7, KG3056 (PAO1 type-B nfxB); 8, KG2236 (KG3056 mexR::res-Ω); and 9, KG2259 (KG3056 ΔmexR-mexA-mexB-oprM).