Date Published: October 5, 2018
Publisher: Public Library of Science
Author(s): Michael Fruci, Keith Poole, Marie-Joelle Virolle.
Exposure of P. aeruginosa to the aminoglycoside (AG) paromomycin (PAR) induced expression of the PA3720-armR locus and the mexAB-oprM multidrug efflux operon that AmgR controls, although PAR induction of mexAB-oprM was independent of armR. Multiple AGs promoted mexAB-oprM expression and this was lost in the absence of the amgRS locus encoding an aminoglycoside-activated envelope stress-responsive 2-component system (TCS). Purified AmgR bound to the mexAB-oprM promoter region consistent with this response regulator directly regulating expression of the efflux operon. The thiol-active reagent, diamide, which, like AGs, promotes protein aggregation and cytoplasmic membrane damage also promoted AmgRS-dependent mexAB-oprM expression, a clear indication that the MexAB-OprM efflux system is recruited in response to membrane perturbation and/or circumstances that lead to this. Despite the AG and diamide induction of mexAB-oprM, however, MexAB-OprM does not appear to contribute to resistance to these agents.
Pseudomonas aeruginosa is a common nosocomial human pathogen [1, 2] often associated with pulmonary infections in patients with cystic fibrosis . The organism has an impressive intrinsic antimicrobial resistome  and readily develops resistance during antimicrobial therapy via mutation and horizontal gene transfer [5–7]. Major contributors to intrinsic and acquired antimicrobial resistance in P. aeruginosa are a number of broadly-specific multidrug efflux systems of the RND family . One of these, MexAB-OprM, which contributes to both intrinsic  and acquired (i.e., mutational)  resistance, exhibits one of the broadest substrate profiles of the RND pumps in P. aeruginosa, accommodating a wide range of clinically-relevant [10–12] and experimental  antimicrobials as well as biocides  and a variety of non-clinical agents (e.g., organic solvents , dyes [16, 17], detergents , herbicides  and acylhomoserine lactones (AHLs) associated with quorum-sensing (QS) . Clinically, this efflux system is most noted for its contribution to acquired fluoroquinolone and β-lactam resistance .
AmgR represents yet another direct regulator of the mexAB-oprM multidrug efflux operon, highlighting the complexity of the regulation of this locus and the apparent diversity of signals and growth conditions to which it responds. Given the apparent responsiveness of the AmgRS TCS to envelope stress resultant from cytoplasmic membrane perturbation by AG-generated mistranslation products, it is likely that the observed AmgRS-dependent induction of mexAB-oprM by AGs reflects an envelope stress-inducibility of this efflux locus and, so, a need for MexAB-OprM under certain conditions of envelope stress. Consistent with this, another agent shown here to be membrane perturbing, diamide, also showed AmgRS-dependent induction of mexAB-oprM expression and this TCS appeared to protect P. aeruginosa somewhat from diamide-mediated membrane damage. While CM perturbation is not a heretofore reported property of diamide, it has been shown that a mutant of Xanthamonas campestris pv. campestris lacking the RpoE envelope stress response sigma is more sensitive to diamide than WT X. campestris , further support for this agent being membrane-damaging. It is interesting to note, too, that the AG induction of mexAB-oprM was variable and reflected the AG responsiveness of AmgRS  with PAR and NEO being the better inducers/activators while, for example, TOB failed to induce/activate (at 1X MIC) and, indeed, perturb membranes . Still, despite the induction of this multidrug efflux locus by AGs and diamide, MexAB-OprM does not appear to play any role in resistance to these agents. It is also possible that AG induction of mexAB-oprM serves primarily to increase OprM levels, this outer membrane protein functioning with the proteins products of the similarly AmgRS-regulated and AG-inducible mexXY efflux operon, with MexXY-OprM a known contributor to AG resistance. Still, oprM appears to possess its own promoter , such that its expression can be driven independently of mexAB, to ensure, for example, adequate levels of OprM to partner with MexXY without the need for unnecessary and wasteful induction of the mexAB genes.