scholarly journals Role of AxyZ Transcriptional Regulator in Overproduction of AxyXY-OprZ Multidrug Efflux System in Achromobacter Species Mutants Selected by Tobramycin

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Julien Bador ◽  
Catherine Neuwirth ◽  
Nadège Grangier ◽  
Marie Muniz ◽  
Leslie Germé ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Transcriptional Regulator ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Resistance Level ◽  
Content Type ◽  
Multidrug Efflux System

ABSTRACT AxyXY-OprZ is an RND-type efflux system that confers innate aminoglycoside resistance to Achromobacter spp. We investigated here a putative TetR family transcriptional regulator encoded by the axyZ gene located upstream of axyXY-oprZ. An in-frame axyZ gene deletion assay led to increased MICs of antibiotic substrates of the efflux system, including aminoglycosides, cefepime, fluoroquinolones, tetracyclines, and erythromycin, indicating that the product of axyZ negatively regulates expression of axyXY-oprZ. Moreover, we identified an amino acid substitution at position 29 of AxyZ (V29G) in a clinical Achromobacter strain that occurred during the course of chronic respiratory tract colonization in a cystic fibrosis (CF) patient. This substitution, also detected in three other strains exposed in vitro to tobramycin, led to an increase in the axyY transcription level (5- to 17-fold) together with an increase in antibiotic resistance level. This overproduction of AxyXY-OprZ is the first description of antibiotic resistance acquisition due to modification of a chromosomally encoded mechanism in Achromobacter and might have an impact on the management of infected CF patients. Indeed, tobramycin is widely used for aerosol therapy within this population, and we have demonstrated that it easily selects mutants with increased MICs of not only aminoglycosides but also fluoroquinolones, cefepime, and tetracyclines.

scholarly journals Oxidative Stress Induction of the MexXY Multidrug Efflux Genes and Promotion of Aminoglycoside Resistance Development inPseudomonas aeruginosa

2010 ◽  
Vol 55 (3) ◽  
pp. 1068-1074 ◽  
Author(s):  
Sebastien Fraud ◽  
Keith Poole
Keyword(s):  
Multidrug Efflux ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Resistance Development ◽  
Enhanced Resistance ◽  
Resistant Mutants ◽  
Multidrug Efflux System

ABSTRACTExposure 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 inPseudomonas aeruginosa. mexXYwas also induced by peroxide exposure, and this too was PA5471 dependent. The prospect of ROS promotingmexXYexpression and aminoglycoside resistance recallsP. aeruginosainfection 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 resistancein vitro, long-term (8-day) exposure ofP. aeruginosato peroxide (mimicking chronicin vivoROS exposure) increased aminoglycoside resistance frequency, dependent upon PA5471 andmexXY. 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, withmexXYgenerally required for this resistance. Moreover, PA5471 was required formexXYexpression and aminoglycoside resistance in these as well as several CF isolates examined.

scholarly journals Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Paulo Juarez ◽  
Katy Jeannot ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Redox Homeostasis ◽  
Transcriptional Regulator ◽  
Significant Loss ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Constitutive Overexpression

ABSTRACT The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa. However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN. PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli. Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

scholarly journals Mutations in Ribosomal Protein RplA or Treatment with Ribosomal Acting Antibiotics Activates Production of Aminoglycoside Efflux Pump SmeYZ in Stenotrophomonas maltophilia

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Karina Calvopiña ◽  
Punyawee Dulyayangkul ◽  
Matthew B. Avison
Keyword(s):  
Ribosomal Protein ◽  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Clinical Isolates ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Ribosomal Protein L1 ◽  
Significant Mechanism

ABSTRACT Aminoglycoside resistance in Stenotrophomonas maltophilia is multifactorial, but the most significant mechanism is overproduction of the SmeYZ efflux system. By studying laboratory-selected mutants and clinical isolates, we show here that damage to the 50S ribosomal protein L1 (RplA) activates SmeYZ production. We also show that gentamicin and minocycline, which target the ribosome, induce expression of smeYZ. These findings explain the role of SmeYZ in both intrinsic and mutationally acquired aminoglycoside resistance.

scholarly journals Polymyxin Susceptibility in Pseudomonas aeruginosa Linked to the MexXY-OprM Multidrug Efflux System

2015 ◽  
Vol 59 (12) ◽  
pp. 7276-7289 ◽  
Author(s):  
Keith Poole ◽  
Calvin Ho-Fung Lau ◽  
Christie Gilmour ◽  
Youai Hao ◽  
Joseph S. Lam
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistance Mechanism ◽  
Polymyxin B ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Content Type ◽  
Polymyxin E ◽  
Increased Susceptibility ◽  
Multidrug Efflux System

ABSTRACTThe ribosome-targeting antimicrobial, spectinomycin (SPC), strongly induced themexXYgenes of the MexXY-OprM multidrug efflux system inPseudomonas aeruginosaand increased susceptibility to the polycationic antimicrobials polymyxin B and polymyxin E, concomitant with a decrease in expression of the polymyxin resistance-promoting lipopolysaccharide (LPS) modification loci,arnBCADTEFand PA4773-74. Consistent with the SPC-promoted reduction inarnand PA4773-74 expression being linked tomexXY, expression of these LPS modification loci was moderated in a mutant constitutively expressingmexXYand enhanced in a mutant lacking the efflux genes. Still, the SPC-mediated increase in polymyxin susceptibility was retained in mutants lackingarnBand/or PA4773-74, an indication that their reduced expression in SPC-treated cells does not explain the enhanced polymyxin susceptibility. That the polymyxin susceptibility of a mutant strain lackingmexXYwas unaffected by SPC exposure, however, was an indication that the unknown polymyxin resistance ‘mechanism’ is also influenced by the MexXY status of the cell. In agreement with SPC and MexXY influencing polymyxin susceptibility as a result of changes in the LPS target of these agents, SPC treatment yielded a decline in common polysaccharide antigen (CPA) synthesis in wild-typeP. aeruginosabut not in the ΔmexXYmutant. A mutant lacking CPA still showed the SPC-mediated decline in polymyxin MICs, however, indicating that the loss of CPA did not explain the SPC-mediated MexXY-dependent increase in polymyxin susceptibility. It is possible, therefore, that some additional change in LPS promoted by SPC-inducedmexXYexpression impacted CPA synthesis or its incorporation into LPS and that this was responsible for the observed changes in polymyxin susceptibility.

scholarly journals Constitutive Activation of MexT by Amino Acid Substitutions Results in MexEF-OprN Overproduction in Clinical Isolates ofPseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Paulo Juarez ◽  
Isabelle Broutin ◽  
Christophe Bordi ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Content Type ◽  
Constitutive Activation ◽  
Multidrug Efflux System

ABSTRACTWhen overproduced, the multidrug efflux system MexEF-OprN increases the resistance ofPseudomonas aeruginosato fluoroquinolones, chloramphenicol, and trimethoprim. In this work, we demonstrate that gain-of-function mutations in the regulatory genemexTresult in oligomerization of the LysR regulator MexT, constitutive upregulation of the efflux pump, and increased resistance in clinical isolates.

scholarly journals Role of the MexAB-OprM Efflux Pump of Pseudomonas aeruginosa in Tolerance to Tea Tree (Melaleuca alternifolia) Oil and Its Monoterpene Components Terpinen-4-ol, 1,8-Cineole, and α-Terpineol

2008 ◽  
Vol 74 (6) ◽  
pp. 1932-1935 ◽  
Author(s):  
Chelsea J. Papadopoulos ◽  
Christine F. Carson ◽  
Barbara J. Chang ◽  
Thomas V. Riley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agent ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Melaleuca Alternifolia ◽  
Tea Tree ◽  
Multidrug Efflux System

ABSTRACT Using a series of efflux mutants of Pseudomonas aeruginosa, the MexAB-OprM pump was identified as contributing to this organism's tolerance to the antimicrobial agent tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and α-terpineol. These data show that a multidrug efflux system of P. aeruginosa can extrude monoterpenes and related alcohols.

scholarly journals The BpeAB-OprB Efflux Pump of Burkholderia pseudomallei 1026b Does Not Play a Role in Quorum Sensing, Virulence Factor Production, or Extrusion of Aminoglycosides but Is a Broad-Spectrum Drug Efflux System

2010 ◽  
Vol 54 (8) ◽  
pp. 3113-3120 ◽  
Author(s):  
Takehiko Mima ◽  
Herbert P. Schweizer
Keyword(s):  
Quorum Sensing ◽  
Virulence Factor ◽  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Homoserine Lactone ◽  
Genetically Engineered ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Aminoglycoside Resistance

ABSTRACT Most Burkholderia pseudomallei strains are intrinsically aminoglycoside resistant, mainly due to AmrAB-OprA-mediated efflux. Rare naturally occurring or genetically engineered mutants lacking this pump are aminoglycoside susceptible despite the fact that they also encode and express BpeAB-OprB, which was reported to mediate efflux of aminoglycosides in the Singapore strain KHW. To reassess the role of BpeAB-OprB in B. pseudomallei aminoglycoside resistance, we used mutants overexpressing or lacking this pump in either AmrAB-OprA-proficient or -deficient strain 1026b backgrounds. Our data show that BpeAB-OprB does not mediate efflux of aminoglycosides but is a multidrug efflux system which extrudes macrolides, fluoroquinolones, tetracyclines, acriflavine, and, to a lesser extent, chloramphenicol. Phylogenetically, BpeAB-OprB is closely related to Pseudomonas aeruginosa MexAB-OprM, which has a similar substrate spectrum. AmrAB-OprA is most closely related to MexXY, the only P. aeruginosa efflux pump known to extrude aminoglycosides. Since BpeAB-OprB in strain KHW was also implicated in playing a major role in export of acylated homoserine lactone (AHL) quorum-sensing molecules and in expression of diverse virulence factors, we explored whether this was also true in the strain 1026b background. The results showed that BpeAB-OprB was not required for AHL export, and mutants lacking this efflux system exhibited normal swimming motility and siderophore production, which were severely impaired in KHW bpeAB-oprB mutants. Biofilm formation was impaired in 1026b Δ(amrRAB-oprA) and Δ(amrRAB-oprA) Δ(bpeAB-oprB) mutants. At present, we do not know why our BpeAB-OprB susceptibility and virulence factor expression results with 1026b and its derivatives are different from those previously published for Singapore strain KHW.

scholarly journals Switch-Loop Flexibility Affects Transport of Large Drugs by the Promiscuous AcrB Multidrug Efflux Transporter

2014 ◽  
Vol 58 (8) ◽  
pp. 4767-4772 ◽  
Author(s):  
Hi-jea Cha ◽  
Reinke T. Müller ◽  
Klaas M. Pos
Keyword(s):  
Local Effect ◽  
Drug Binding ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Minimal Projection ◽  
Content Type ◽  
Binding Pockets ◽  
Loop Flexibility ◽  
Multidrug Efflux System

ABSTRACTMultidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For theresistancenodulation and celldivision (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system fromEscherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å2are less well transported than other substrates.

scholarly journals Reciprocal Regulation of PASTA Kinase Signaling by Differential Modification

2019 ◽  
Vol 201 (10) ◽  
Author(s):  
Benjamin D. Labbe ◽  
Cherisse L. Hall ◽  
Stephanie L. Kellogg ◽  
Yao Chen ◽  
Olivia Koehn ◽  
...  
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Kinase Activity ◽  
Cell Envelope ◽  
Activation Loop ◽  
Intrinsic Resistance ◽  
Content Type

ABSTRACTTransmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP]andSer/Thr-associated) domains are ubiquitous amongActinobacteriaandFirmicutesspecies. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylationin vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood.Enterococcus faecalisis a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. InE. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreKin vivo. Here we identify multiple sites of phosphorylation on IreK and evaluate their functionin vivoandin vitro. While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activityin vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreKin vivoupon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activityin vivoto promote adaptation to cell wall stresses.IMPORTANCETransmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous amongActinobacteriaandFirmicutesspecies and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase ofEnterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistancein vivo. Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

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