Mutations in PA2491 ( mexS ) Promote MexT-Dependent mexEF-oprN Expression and Multidrug Resistance in a Clinical Strain of Pseudomonas aeruginosa

Abstract

Disruption of the PA2491 gene in a mini-Tn5-tet insertion mutant of a clinical isolate of Pseudomonas aeruginosa increased expression of the mexEF-oprN multidrug efflux genes and decreased production of outer membrane protein OprD, concomitant with enhanced resistance to chloramphenicol, quinolones, and imipenem, which was reminiscent of previously described nfxC mutants. PA2491 encodes a probable oxidoreductase previously shown to be positively regulated by the MexT positive regulator of mexEF-oprN expression (T. Köhler, S. F. Epp, L. K. Curty, and J. C. Pechére, J. Bacteriol. 181:6300-6305, 1999). Spontaneous multidrugresistant mutants of the P. aeruginosa clinical isolate hyperexpressing mexEF-oprN and showing reduced production of OprD were readily selected in vitro, and all of them were shown to carry mutations in PA2491, highlighting the probable significance of such mutations as determinants of MexEF-OprN-mediated multidrug resistance in vivo.Three-component multidrug efflux systems of the resistancenodulation-division (RND) family are prevalent in gram-negative bacteria, in which they contribute significantly to intrinsic and acquired resistance to a range of clinically important antimicrobial agents (i.e., antibiotics and biocides) (48). In Pseudomonas aeruginosa, an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents (19), seven tripartite RND family efflux systems have been described to date 35,50,51 [25,52,54,64,73] and mexZ [40,69], respectively).The MexEF-OprN system is apparently quiescent in wildtype cells, at least under the usual laboratory growth conditions (32), but it is expressed in nfxC-type multidrug-resistant strains isolated in vitro (14, 32, 42) and in clinics (15,24). nfxC mutants display resistance to fluoroquinolones, chloramphenicol, trimethoprim, and the carbapenem imipenem (14, 32), although resistance to imipenem results not from MexEF-OprN expression (32) but from the concomitant decrease in outer membrane protein OprD in these mutants (14,42). OprD is a basic amino acid-peptide channel and a primary route of entry for carbapenems, such as imipenem, in P. aeruginosa (67, 68), and it is often absent in imipenem-resistant strains of P. aeruginosa (4, 31). In addition to its role in the export of and resistance to antimicrobials, MexEF-OprN also promotes resistance (or tolerance) to organic solvents (36), dyes (16), and biocides, such as triclosan (8). As a result of MexEF-OprN hyperexpression, nfxC mutants also express reduced levels of several quorum-sensing (i.e., homoserine lactone)-dependent extracellular virulence factors (33) and are attenuated for virulence (10), apparently as a result of MexEF-OprN-mediated export of a molecule(s) necessary for homoserine lactone production (33).Unlike the majority of RND-type efflux systems in P. aeruginosa, which are negatively regulated by linked repressor genes, MexEF-OprN expression is positively regulated by the product of the linked mexT gene, a LysR family regulator of mexEFoprN expr...

Influence of the regulatory protein RsmA on cellular functions in Pseudomonas aeruginosa PAO1, as revealed by transcriptome analysis

“…Examination of MexT protein levels in the rsmA mutant showed that they were not altered (data not shown). However, levels of mexS mRNA, which encodes a transcriptional repressor of MexEF-OprN, (Sobel et al, 2005) were increased fourfold in the rsmA mutant compared to PAO1. These results reflect the complexity of Mex pump regulation.…”

Influence of Quorum Sensing and Iron on Twitching Motility and Biofilm Formation in Pseudomonas aeruginosa

“…Plasmid pGP003, carrying the rhlI gene, was constructed by amplification of rhlI from the chromosome of P. aeruginosa K1120 by PCR using primers rhlI-F (5Ј-GGATCCGGATCCCTGCAATGGACCGCCGAC-3Ј; tandem BamHI sites underlined) and rhlI-R (5Ј-AAGCTTAAGCTTGCATGCGG CAGG-AGAAAGC-3Ј; tandem HindIII sites underlined) and cloning the gene into pMMB207. Amplification was carried out in 50-l reaction mixtures formulated as described previously, using Vent DNA polymerase (New England Biolabs) (79), and subjected to a 5-min denaturation step at 95°C, followed by 30 cycles of 5 min at 95°C, 1 min at 61.8°C (annealing temperature), and 5 min at 95°C, before finishing with 10 min at 72°C. To introduce a deletion of the rhlR gene into P. aeruginosa K1120, a deletion construct was first prepared in plasmid pEX18Tc by cloning PCR-amplified 1-kb DNA fragments corresponding to the regions upstream and downstream of the rhlR sequences to be deleted.…”

Citrate-mediated iron uptake in Pseudomonas aeruginosa: involvement of the citrate-inducible FecA receptor and the FeoB ferrous iron transporter

“…The pyoverdine-and pyochelin-deficient P. aeruginosa strain, IA614, was mutagenized with mini-Tn5-tet (de Lorenzo et al, 1990) following mobilization of mini-Tn5-tetcarrying plasmid pUT from E. coli SM10 (l pir ) as described previously (Sobel et al, 2005), with the IA614 mini-Tn5-tet insertion mutants selected on L-agar plates containing tetracycline (60 mg ml 21 ) and imipenem (0.5 mg ml 21 ; to counter-select donor E. coli). To screen for mutants with defects in citrate-mediated iron acquisition, two approaches were taken.…”