Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-oprM multidrug efflux system of Pseudomonas aeruginosa

Several nalB-type multidrug-resistant mutants of Pseudomonas aeruginosa overexpressed MexAB-OprM and carried mutations in the local regulatory gene, mexR. Others, dubbed nalC types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the nalB strains. Available evidence showed that MexR acted solely as repressor. Disruption of the mexR gene at various places suggested that the 5' end of mexR may be a part of the mexAB-oprM promoter.     

The MexA-MexB-OprM multidrug efflux system of Pseudomonas aeruginosa is growth-phase regulated

Intrinsic antibiotic resistance in Pseudomonas aeruginosa is attributed to low outer membrane permeability and drug efflux mediated by the products of mexAmexBoprM efflux operon. Using a mexA-phoA fusion, expression of the efflux genes was assessed as a function of growth in a variety of strains. The efflux operon was growth-phase regulated in both wild-type and nalB strains, being minimally expressed in lag phase and increasing in log to late log phase. MexR, the only known regulator of MexAMexBOprM and target of mutation in nalB strains, was not involved in the growth-phase regulation. The las cascade regulates genes based on increased cell-density, but a deletion in lasR had no effect on mexAmexBoprM expression. Putative recognition sequences for AlgT/U and RpoN were identified upstream of mexA, but algT/U and rpoN null mutants also had no effect on mexAmexBoprM expression.     

MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719

MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.     

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.     

Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.     

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.     

Chlorinated phenols control the expression of the multidrug resistance efflux pump MexAB-OprM in Pseudomonas aeruginosa by interacting with NalC

NalC is a TetR type regulator that represses the multidrug efflux pump MexAB-OprM in Pseudomonas aeruginosa. Here we explain the mechanism of NalC-mediated regulation of MexAB-OprM. We show that NalC non-covalently binds chlorinated phenols and chemicals containing chlorophenol side-chains such as triclosan. NalC-chlorinated phenol binding results in its dissociation from promoter DNA and upregulation of NalC's downstream targets, including the MexR antirepressor ArmR. ArmR upregulation and MexR-ArmR complex formation have previously been shown to upregulate MexAB-OprM. In vivo mexB and armR expression analyses were used to corroborate in vitro NalC-chlorinated phenol binding. We also show that the interaction between chlorinated phenols and NalC is reversible, such that removal of these chemicals restored NalC promoter DNA binding. Thus, the NalC-chlorinated phenol interaction is likely a pertinent physiological mechanism that P. aeruginosa uses to control expression of the MexAB-OprM efflux pump.     

Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa encodes three types of xenobiotic efflux pumps, MexAB-OprM, MexCD-OprJ, and MexEF-OprN, which are regulated by the nalB, nfxB, and nfxC genes, respectively, and their high expression renders the cells resistant to multiple species of antibiotics. We evaluated the role of the outer membrane permeability barrier and the efflux pump in lowering the intracellular concentration of fluorescent probes. The wild-type, nalB, nfxB, and nfxC strains with an intact outer membrane showed equally high capability in draining out intracellular fluorescent dye, 2-(4-dimethylaminostyryl)-1-ethylpyridinium and ethidium bromide. When the outer membrane barrier was dismantled by the EDTA treatment, wild-type, nfxC, nfxB, and nalB strains showed significantly different levels of dye accumulation. The polymyxin B-treated cells showed an even more pronounced difference in dye accumulation among the nfxC, nfxB, and nalB mutants. We concluded from these results that the xenobiotic extrusion pumps interplay with the outer membrane permeability barrier in lowering the intracellular substrate concentration. Among three extrusion pumps in P. aeruginosa, MexAB-OprM was the most efficient, followed by MexCD-OprJ and MexEF-OprN pumps for the fluorescent dye extrusion.     

Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression

We have isolated 25 MexR mutants that retained their dimerizing ability but were unable to bind mexOP DNA. Surprisingly, 20 mutations were located in the hydrophobic core region at alpha4, W1, alpha2, alpha3, and beta2, and only 3 were in positively charged residues. These results verified that DNA binding is mediated by distinct regions of MexR and showed the importance of the hydrophobic core region of the DNA-binding domain.     

Differential effects of mutations in tonB1 on intrinsic multidrug resistance and iron acquisition in Pseudomonas aeruginosa

Loss of tonB1 adversely affects iron acquisition and intrinsic multidrug resistance in Pseudomonas aeruginosa. Several mutations in tonB1 compromised the protein's contribution to both processes, although TonB1 derivatives altered in residues C35, Q268, R287, Q292, R300, and R304 were compromised vis-à-vis their contribution to drug resistance only.     

Mutation in mexR-gene leading to drug resistance in corneal keratitis in human

The present study deals with the genetic polymorphism of the mexR gene which is involved in the resistance to drugs like ciprofloxacin. Mutations in mexR result in increased resistance to multiple antibiotics due to overexpression of this efflux system. The MexR product contains 147 amino acids with a molecular mass of 16,964 Da. We detected 28 point mutations in 14 samples from corneal scraping infected with Pseudomonas aeruginosa, which were screened for ciprofloxacin resistance. Twenty four were silent mutations and four missense mutations. Mapping these mutations was done by using in silico methods on the protein 3D- structure obtained from PDB database, localized at 3 specific sites. Single amino acid changes (mutations) may influence MexR stability or its ability to dimerise, and thus result in the conformation changes at the DNA-binding domain, of the structure. Hence it is concluded that the mutations in the DNA binding domain of mexR gene could be one of the factors contributing to the possible drug resistance in these patients.