Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response

Balasubramanian, Deepak; Kumari, Hansi; Jaric, Melita; Fernandez, Mitch; Turner, Keith H.; Dove, Simon L.; Narasimhan, Giri; Lory, Stephen; Mathee, Kalai

doi:10.1093/nar/gkt942

Cited by 54 publications

(92 citation statements)

References 132 publications

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“…This is in accordance with the current model where, in the absence of ␤-lactams, AmpR represses ampC transcription, and when exposed to antibiotics, cells accumulate peptidoglycan catabolites (i.e., 1,6-anhydroMurNAcpeptides), changing AmpR to an activator of ampC transcription (33,46,47). In P. aeruginosa, AmpR plays a role in physiological processes and influences the expression of over 500 genes, including virulence-encoding genes and other transcriptional regulators (15,16,48). Then, AmpR appears as a global regulator, and its possible role in the virulence of ECC should be further investigated.…”

Section: Resultssupporting

confidence: 90%

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Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

Guérin

Isnard

Cattoir

et al. 2015

Antimicrob Agents Chemother

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bEnterobacter cloacae complex (ECC), an opportunistic pathogen causing numerous infections in hospitalized patients worldwide, is able to resist ␤-lactams mainly by producing the AmpC ␤-lactamase enzyme. AmpC expression is highly inducible in the presence of some ␤-lactams, but the underlying genetic regulation, which is intricately linked to peptidoglycan recycling, is still poorly understood. In this study, we constructed different mutant strains that were affected in genes encoding enzymes suspected to be involved in this pathway. As expected, the inactivation of ampC, ampR (which encodes the regulator protein of ampC), and ampG (encoding a permease) abolished ␤-lactam resistance. Reverse transcription-quantitative PCR (qRT-PCR) experiments combined with phenotypic studies showed that cefotaxime (at high concentrations) and cefoxitin induced the expression of ampC in different ways: one involving NagZ (a N-acetyl-␤-D-glucosaminidase) and another independent of NagZ. Unlike the model established for Pseudomonas aeruginosa, inactivation of DacB (also known as PBP4) was not responsible for a constitutive ampC overexpression in ECC, whereas it caused AmpC-mediated high-level ␤-lactam resistance, suggesting a posttranscriptional regulation mechanism. Global transcriptomic analysis by transcriptome sequencing (RNA-seq) of a dacB deletion mutant confirmed these results. Lastly, analysis of 37 ECC clinical isolates showed that amino acid changes in the AmpD sequence were likely the most crucial event involved in the development of high-level ␤-lactam resistance in vivo as opposed to P. aeruginosa where dacB mutations have been commonly found. These findings bring new elements for a better understanding of ␤-lactam resistance in ECC, which is essential for the identification of novel potential drug targets.

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Section: Resultssupporting

confidence: 90%

“…In Pseudomonas aeruginosa, several works showed that AmpR is a global transcriptional regulator. It is a potential membrane-associated dimer that regulates genes involved in virulence, quorum sensing, and stress response (14)(15)(16)(17)(18). In addition, three AmpD enzymes have been found in P. aeruginosa.…”

mentioning

confidence: 99%

Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

Guérin

Isnard

Cattoir

et al. 2015

Antimicrob Agents Chemother

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“…Moreover, a recent work suggested that the activation of the nitrate respiratory chain in P. aeruginosa might be a response in order to compensate for the fitness cost associated with particular antibiotic resistance mechanisms (50). Therefore, our results complement previous studies performed with the AmpR mutant that point out a link between ␤-lactamase and global regulation in P. aeruginosa (3,51,52). Indeed, although the exact CreBC regulatory mechanisms are still unknown, we demonstrate the complexity and multiregulatory processes by which P. aeruginosa controls the expression of genes of diverse functions from ␤-lactam resistance to biofilm formation or anaerobic respiration.…”

Section: Resultssupporting

confidence: 89%

The Pseudomonas aeruginosa CreBC Two-Component System Plays a Major Role in the Response to β-Lactams, Fitness, Biofilm Growth, and Global Regulation

Zamorano

Moyá

Juan

et al. 2014

Antimicrob Agents Chemother

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bPseudomonas aeruginosa is a ubiquitous versatile environmental microorganism with a remarkable ability to grow under diverse environmental conditions. Moreover, P. aeruginosa is responsible for life-threatening infections in immunocompromised and cystic fibrosis patients, as the extraordinary capacity of this pathogen to develop antimicrobial resistance dramatically limits our therapeutic arsenal. Its large genome carries an outstanding number of genes belonging to regulatory systems, including multiple two-component sensor-regulator systems that modulate the response to the different environmental stimuli. Here, we show that one of two systems, designated CreBC (carbon source responsive) and BlrAB (␤-lactam resistance), might be of particular relevance. We first identified the stimuli triggering the activation of the CreBC system, which specifically responds to penicillin-binding protein 4 (PBP4) inhibition by certain ␤-lactam antibiotics. Second, through an analysis of a large comprehensive collection of mutants, we demonstrate an intricate interconnection between the CreBC system, the peptidoglycan recycling pathway, and the expression of the concerning chromosomal ␤-lactamase AmpC. Third, we show that the CreBC system, and particularly its effector inner membrane protein CreD, plays a major role in bacterial fitness and biofilm development, especially in the presence of subinhibitory concentrations of ␤-lactams. Finally, global transcriptomics reveals broad regulatory functions of CreBC in basic physiological aspects, particularly anaerobic respiration, in both the presence and absence of antibiotics. Therefore, the CreBC system is envisaged as a potentially interesting target for improving the efficacy of ␤-lactams against P. aeruginosa infections.

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“…The role of AmpR as regulator of ampC expression has been clearly established in both the Enterobacteriaceae and P. aeruginosa. Our recent work has further redefined AmpR as a major global regulator, playing an important role in acute infections through its regulation of virulence, biofilm formation, quorum sensing, and non-␤-lactam resistance (31,(39)(40)(41)87). Regulation of ampC, however, remains one of its most critical roles, as AmpC derepression is a prevalent mechanism of ␤-lactam resistance in P. aeruginosa.…”

Section: Fig 8 Stability Of P Aeruginosa Ampr Mutant Proteins Totalmentioning

confidence: 99%

Structural and Functional Characterization of Pseudomonas aeruginosa Global Regulator AmpR

et al. 2014

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Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC ␤-lactamase is a common mechanism of ␤-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (P ampC ) and ampR (P ampR ) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5= rapid amplification of cDNA ends-PCR (RACE-PCR), and strong 54 and 70 consensus sequences were identified at P ampR and P ampC , respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal P ampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to P ampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding.

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Deep sequencing analyses expands the Pseudomonas aeruginosa AmpR regulon to include small RNA-mediated regulation of iron acquisition, heat shock and oxidative stress response

Cited by 54 publications

References 132 publications

Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

Complex Regulation Pathways of AmpC-Mediated β-Lactam Resistance in Enterobacter cloacae Complex

The Pseudomonas aeruginosa CreBC Two-Component System Plays a Major Role in the Response to β-Lactams, Fitness, Biofilm Growth, and Global Regulation

Structural and Functional Characterization of Pseudomonas aeruginosa Global Regulator AmpR

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