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Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa
T Köhler1, M Michéa-Hamzehpour, U Henze
1Department of Genetics and Microbiology, Centre Médical Universitaire, Geneva, Switzerland. Kohler@cmu.unige.ch
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.
Insights
Pseudomonas aeruginosa mutants developed multidrug resistance via the MexEF-OprN efflux system. This system, regulated by a LysR-family activator, also aids in pyocyanin excretion.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas aeruginosa is an opportunistic pathogen known for its intrinsic and acquired antibiotic resistance.
- Multidrug resistance (MDR) in P. aeruginosa is often mediated by efflux pumps, particularly those belonging to the RND (Resistance-Nodulation-Division) superfamily.
- Understanding novel efflux systems is crucial for combating antibiotic resistance in clinical settings.
Purpose of the Study:
- To identify and characterize a novel multidrug efflux system in Pseudomonas aeruginosa.
- To elucidate the genetic basis and regulatory mechanisms of this efflux system.
- To investigate the potential role of this system in the excretion of secondary metabolites.
Main Methods:
- Generation and characterization of antibiotic-resistant mutants of P. aeruginosa.
- Outer membrane protein analysis using Western blotting.
- Cosmid complementation assays to isolate the relevant gene.
- Antibiotic accumulation studies to assess efflux activity.
- DNA sequencing and bioinformatics analysis to identify open reading frames (ORFs).
- Gene mutation studies to determine the role of specific genes in resistance.
- Analysis of transcriptional regulation using DNA fragments and reporter assays.
Main Results:
- Antibiotic-resistant mutants overexpressed a 50 kDa outer membrane protein, identified as OprN.
- OprN was found to be part of an energy-dependent antibiotic-efflux system.
- Sequencing revealed three ORFs (MexE, MexF, OprN) homologous to known efflux operon components.
- Mutation of the mexE gene abolished the MDR phenotype in OprN-overexpressing strains.
- A LysR-family transcriptional activator positively regulated the mexEF-oprN operon.
- Expression of the mexEF-oprN operon conferred MDR to wild-type strains but not to mexE mutants.
- The mexEF-oprN operon appears to be involved in the excretion of pyocyanin biosynthesis intermediates.
Conclusions:
- The mexEF-oprN operon constitutes a novel multidrug efflux system in P. aeruginosa.
- MexE, MexF, and OprN are key components of this MDR system.
- The operon is positively regulated by a LysR-family transcriptional activator.
- This efflux system plays a role in both antibiotic resistance and the excretion of pyocyanin.
- Targeting the mexEF-oprN operon could be a strategy to overcome MDR in P. aeruginosa infections.