Oxidative Stress Induction of the MexXY Multidrug Efflux Genes and Promotion of Aminoglycoside Resistance Development in Pseudomonas aeruginosa ▿

Exposure to reactive oxygen species (ROS) (e.g., peroxide) was shown to induce expression of the PA5471 gene, which was previously shown to be required for antimicrobial induction of the MexXY components of the MexXY-OprM multidrug efflux system and aminoglycoside resistance determinant in Pseudomonas aeruginosa. mexXY was also induced by peroxide exposure, and this too was PA5471 dependent. The prospect of ROS promoting mexXY expression and aminoglycoside resistance recalls P. aeruginosa infection of the chronically inflamed lungs of cystic fibrosis (CF) patients, where the organism is exposed to ROS and where MexXY-OprM predominates as the mechanism of aminoglycoside resistance. While ROS did not enhance aminoglycoside resistance in vitro, long-term (8-day) exposure of P. aeruginosa to peroxide (mimicking chronic in vivo ROS exposure) increased aminoglycoside resistance frequency, dependent upon PA5471 and mexXY. This enhanced resistance frequency was also seen in a mutant strain overexpressing PA5471, in the absence of peroxide, suggesting that induction of PA5471 by peroxide was key to peroxide enhancement of aminoglycoside resistance frequency. Resistant mutants selected following peroxide exposure were typically pan-aminoglycoside-resistant, with mexXY generally required for this resistance. Moreover, PA5471 was required for mexXY expression and aminoglycoside resistance in these as well as several CF isolates examined

Antibiotic inducibility of the mexXY multidrug efflux operon of Pseudomonas aeruginosa: involvement of the MexZ anti-repressor ArmZ.

Expression of the mexXY multidrug efflux operon in wild type Pseudomonas aeruginosa is substantially enhanced by the ribosome-targeting antimicrobial spectinomycin (18-fold) and this is wholly dependent upon the product of the PA5471 gene. In a mutant strain lacking the mexZ gene encoding a repressor of mexXY gene expression, expression of the efflux operon increases modestly (5-fold) and is still responsive (18-fold) to spectinomycin. Spectinomycin induction of mexXY expression in the mexZ mutant is, however, independent of PA5471 suggesting that PA5471 functions as an anti-repressor (dubbed ArmZ for anti-repressor MexZ) that serves only to modulate MexZ's repressor activity, with additional gene(s)/gene product(s) providing for the bulk of the antimicrobial-inducible mexXY expression. Consistent with PA5471/ArmZ functioning as a MexZ anti-repressor, an interaction between MexZ and ArmZ was confirmed using a bacterial 2-hybrid assay. Mutations compromising this interaction (P68S, G76S, R216C, R221W, R221Q, G231D and G252S) were identified and localized to one region of an ArmZ structural model that may represent a MexZ-interacting domain. Introduction of representative mutations into the chromosome of P. aeruginosa reduced (P68S, G76S) or obviated (R216C, R2211W) antimicrobial induction of mexXY gene expression, rendering the mutants pan-aminoglycoside-susceptible. These data confirm the importance of an ArmZ-MexZ interaction for antimicrobial-inducible mexXY expression and intrinsic aminoglycoside resistance in P. aeruginosa

The effect of reduced sodium chloride content on the development of fluorescent Pseudomonas in a Reblochon-like cheese

The effect of reduced sodium chloride content on the development of fluorescent Pseudomonas in a Reblochon-like cheese. Microbial Spoilers in Food 201

Bacterial strains and plasmids.

a<p>Tc^r, tetracycline resistance; Ap^r, ampicillin resistance.</p>b<p>The amino acid substitutions in the ArmZ products produced by the indicated <i>armZ</i> mutant strains are highlighted in parentheses.</p>c<p>The amino acid substitutions in the mutant ArmZ products encoded by the indicated plasmids are highlighted in parentheses. WT, wild type.</p

Influence of ArmZ on spectinomycin-inducible <i>mexXY</i> expression.

<p><i>mexX</i> expression was assessed in the indicated <i>P. aeruginosa</i> strains in the absence (−) or presence (+) of spectinomycin (SPC) using quantitative RT-PCR. The <i>armZ</i> and <i>mexZ</i> status (−, absent; +, present) of the strains is highlighted. Expression was normalized to <i>rpoD</i> and is reported relative (fold change) to the wild-type <i>P. aeruginosa</i> PAO1 strain K767 not exposed to spectinomycin. Values represent the mean ± SEM from at least three independent determinations, each performed in triplicate.</p

Location of mutations in ArmZ that compromise its interaction with MexZ.

<p>The 3-dimensional model of ArmZ was created by threading the ArmZ amino acid sequence onto the crystal structure of the PH1602-extein protein from <i>Pyrococcus horikoshii</i> (PDB code: 1UC2, chain A).</p

MexZ-ArmZ interaction.

<p>A MexZ-ArmZ interaction and the impact of <i>armZ</i> mutations this interaction was assessed using a bacterial 2-hybrid assay, in which the interaction leads to repression of <i>lacZ</i> expression and reduced β-galactosidase activity in the reporter <i>E. coli</i> strain SU202. (a) The β-galactosidase activity of <i>E. coli</i> SU202 harbouring pMS604 derivatives expressing wild type (WT) or no (−) MexZ together with DP804 derivatives expressing wild type (WT), mutant (amino acid substitutions highlighted) or no (−) ArmZ is indicated. The results shown are mean ± SEM from at least three independent determinations, each performed in triplicate. (b) Western immunoblot of whole cell extracts of <i>E. coli</i> SU202 harbouring pMS604 derivatives expressing wild type MexZ and pDP804 derivatives expressing wild type and mutant ArmZ developed with anti-LexA antibodies.</p