Overexpression of the mexC–mexD–oprJ efflux operon in nfxB‐type multidrug‐resistant strains of Pseudomonas aeruginosa

Abstract

OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.

Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa

“…In several clinically isolated mutants of P. aeruginosa, higher expression of RND-type efflux pumps was observed (Hirai et al, 1987;Rella & Haas, 1982;Wolter et al, 2004). Of the 11 RND efflux pumps identified in this organism, 10 pumps, MexAB-OprM (Poole et al, 1993), MexCD-OprJ (Poole et al, 1996), MexEF-OprN (Kohler et al, 1997), MexGHI-OpmD (Aendekerk et al, 2002;Sekiya et al, 2003), MexJK (Chuanchuen et al, 2002), MexMN (Mima et al, 2005), MexPQ-OpmE (Mima et al, 2005), MexVW (Li et al, 2003), MexXY (Mine et al, 1999;Westbrock-Wadman et al, 1999) and TriABCOpmH (Mima et al, 2007), have been experimentally confirmed, and their properties have been reported. The RND-type efflux pump usually consists of three components to fulfil the function properly and includes an innermembrane component (RND component), a periplasmic component (MFP component), and an outer-membrane component (OMP component) (Touze et al, 2004), and the three-dimensional structures of MexA, MexB and OprM have been reported (Akama et al, 2004a, b;Higgins et al, 2004;Sennhauser et al, 2009).…”

Tetracyclines in veterinary medicine and bacterial resistance to them

“…In Pseudomonas aeruginosa, resistance to a variety of antimicrobial agents is conferred by the synergy between low permeability of the outer membrane and several RND multidrug efflux systems, of which MexAB-OprM (Poole, 1993;Li et al, 1994Li et al, , 1995b and MexCD-OprJ (Masuda et al, 1995, Poole et al, 1996 contribute to the resistance to tetracycline. They both consist of three components: MexA or MexC (periplasmic MFP proteins), MexB or MexD (inner membrane RND proteins) and OprM or OprJ (outer membrane channel proteins) and they pump drugs from the cytoplasm into the medium.…”

Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

“…MexC associates with the inner membrane transporter MexD and the outer membrane ef¯ux protein OprJ to form a multidrug export complex, which is overexpressed in strains resistant to chloramphenicol, erythromycin, tetracycline, quinolones, and cefpirom (Miche Âa-Hamzehpour et al, 1995;Poole et al, 1996). The MexC peptides, residues 64-95 and 172-208, completely span the conserved motif (Figure 2).…”