Antibiotic Inducibility of the mexXY Multidrug Efflux Operon of Pseudomonas aeruginosa: Involvement of the MexZ Anti-Repressor ArmZ

Abstract

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.

Resistance suppression by high-intensity, short-duration aminoglycoside exposure against hypermutable and non-hypermutablePseudomonas aeruginosa

“…Common mechanisms involved in the resistance observed in PAO1 could be adaptive resistance via the upregulation of the MexXY-OprM efflux pump, 41,50 which is frequently caused by overexpression of the MexY component, or de novo mutations during treatment. 41,51,52 Previous studies have found that a resistance mechanism unrelated to this efflux pump has played a role in aminoglycoside resistance for PAODmutS. 6 Ultimately, molecular studies would identify these resistance mechanisms.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…5). Their regulators (e.g., MexR, EsrC, ArmZ, and MexT) can respond to various stresses caused by, for example, membrane-damaging or ribosome-disrupting agents, reactive oxygen species, and/or nitrosative stress and subsequently cause increased activities of these pumps against the stresses (441,454,461,467,798,799,827,835,911). Consistently, P. putida RND pump mutants are categorized into 4 functionally distinct subgroups, and one of them is more susceptible to oxidation-inducing agents (912).…”

“…The very low basal expression level of mexXY(oprA) in wild-type bacteria results from the direct binding of the dimerized, strong repressor MexZ to a 20-bp palindromic sequence encompassing the overlapping promoters of mexXY(oprA) and the adjacent, divergently transcribed gene mexZ (458,825,826). Unlike other TetR-type regulators (727), MexZ's DNA binding is not relieved by antibiotics through a direct ligand-regulator interaction but seemingly via indirect protein-protein sequestration, a process relying on the product of the PA5471 gene, named ArmZ, for the antirepressor of mexZ (825)(826)(827). It was demonstrated that the induction of mexXY expression in response to protein synthesis inhibitors is totally dependent upon ArmZ and that the expression of armZ itself is induced by ribosome-targeting agents through a sophisticated mechanism of transcriptional attenuation involving a short 13-amino-acid leader peptide, PA5471.1 (828).…”

“…Thus, the ribosome-perturbing mutations act in a way reminiscent of mexXY induction mediated by ribosome-targeting antibiotics (461). However, effectors other than ArmZ seem to be required for full MexZ-dependent drug induction of the efflux operon, as the latter still remains inducible in mexZ and mexZ-PA5471 knockout mutants (441,827). Furthermore, it was demonstrated that exposure of P. aeruginosa to reactive oxygen species such as H 2 O 2 also results in ArmZ-dependent mexXY(oprA) derepression (454).…”

The TetR Family of Regulators

“…Expression of mexXY is controlled by the TFR MexZ and requires ArmZ (PA5471) (122), which interacts with MexZ (31,123). armZ encodes a homolog of RtcB, an RNA ligase involved in recovery from stress-induced RNA damage (124), and is cotranscribed with PA5470, which encodes a homolog of PrfH.…”