Regulation of the mexAB-oprM efflux operon of Pseudomonas aeruginosa
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Authors
Fruci, Michael
Date
Type
thesis
Language
eng
Keyword
Pseudomonas aeruginosa , MexAB-oprM , Efflux , Multidrug , Resistance-Nodulation-Division , Antimicrobial Resistance , Regulation
Alternative Title
Abstract
Multidrug efflux systems are important determinants of antimicrobial resistance in the human pathogen Pseudomonas aeruginosa where they compromise anti-pseudomonal chemotherapy. The major multidrug efflux system in P. aeruginosa is encoded by the mexAB-oprM operon, expression of which is regulated by a MexR repressor whose activity is negatively modulated by the anti-repressor protein, ArmR. armR occurs as part of a two-gene operon, PA3720-armR, that is regulated by the product of the divergently-transcribed nalC repressor gene, with nalC mutants showing elevated PA3720-armR expression and, so, elevated mexAB-oprM expression and multidrug resistance. The function of the PA3720 protein is unknown. We show here that PA3720 functions as a non-specific RNA binding protein that binds to and destabilizes the adjacent armR-bearing mRNA. Expression of armR is also shown to be translationally coupled to PA3720. In an earlier study, aminoglycosides were shown to induce expression of PA3720-armR, suggesting that these drugs may also promote mexAB-oprM expression dependent on ArmR. Consistent with this, aminoglycosides promoted expression of mexAB-oprM; however, this was independent of ArmR. Instead, the aminoglycoside-responsive AmgRS two-component system mediated aminoglycoside induction of this efflux system. Consistent with this, mutational activation of the AmgS sensor kinase yielded elevated levels of mexAB-oprM expression, and purified AmgR bound specifically to the mexAB-oprM promoter region. Screening of P. aeruginosa strain PAO1 transposon mutants for increased sensitivity to carbenicillin- carbenicillin is a MexAB-OprM substrate and carbenicillin sensitivity is a surrogate for loss of mexAB-oprM expression - led to the identification of several genes with probable roles in mexAB-oprM expression or activity. The gene disrupted in one of these mutants, bamB, encodes for a component of the outer membrane β-barrel protein assembly machinery and was assessed for its role in facilitating the assembly of OprM, a β-barrel outer membrane protein. Western immunoblotting revealed that BamB is not required for OprM insertion into the outer membrane. These studies provide additional insights into the regulation of mexAB-oprM expression in P. aeruginosa.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.