Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa

Abstract

Antibiotic-resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50 kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB-oprM efflux operon. Antibiotic-accumulation studies indicated that OprN was part of an energy-dependent antibiotic-efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB-oprM and mexCD-oprJ efflux operons of P. aeruginosa. The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug-resistance phenotype in an OprN-overexpressing strain, but did not affect the susceptibility profile of the wild-type strain. Expression of the mexEF-oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild-type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF-oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa.

CzcR-CzcS, a Two-component System Involved in Heavy Metal and Carbapenem Resistance in Pseudomonas aeruginosa

“…This type of co-regulation has been described in nfxC type mutants of P. aeruginosa (43). These mutants overexpress the multidrug efflux pump MexEF-OprN (44), conferring resistance to quinolones, chloramphenicol, and trimethoprim, and are resistant to carbapenems due to decreased OprD expression (44,45). Interestingly this resistance phenotype can be obtained in the susceptible wild type PT5 upon plasmid-mediated overexpression of MexT, the transcriptional activator of the MexEF-OprN efflux pump (46,47).…”

“…These mutants overexpress the multidrug efflux pump MexEF-OprN (44), conferring resistance to quinolones, chloramphenicol, and trimethoprim, and are resistant to carbapenems due to decreased OprD expression (44,45). Interestingly this resistance phenotype can be obtained in the susceptible wild type PT5 upon plasmid-mediated overexpression of MexT, the transcriptional activator of the MexEF-OprN efflux pump (46,47). MexT overexpression was shown to decrease both transcriptional (46,47) and posttranscriptional expression of oprD (46).…”

“…Interestingly this resistance phenotype can be obtained in the susceptible wild type PT5 upon plasmid-mediated overexpression of MexT, the transcriptional activator of the MexEF-OprN efflux pump (46,47). MexT overexpression was shown to decrease both transcriptional (46,47) and posttranscriptional expression of oprD (46). Hence both CzcR and MexT are able to repress OprD expression, which could occur either directly or via another regulator (Fig.…”

Amino Acid Residues Essential for Function of the MexF Efflux Pump Protein of Pseudomonas aeruginosa

“…Bacterial strains and plasmids used in this work are listed in Table 1. The P. aeruginosa nfxC-type strain PT644 is a mexB::⍀Hg derivative obtained by transduction of the Hg cassette from the mexB::⍀Hg strain PAM1275 (kindly provided by O. Lomovskaya) into strain PT149 (nfxC) (formerly called PAO-7H) (12). Disruption of the mexF gene in strain PT644 resulted in the ⌬mexF derivative PT644⌬F (see below).…”

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).…”