Characterization of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione resistance in pyomelanogenic Pseudomonas aeruginosa DKN343

Laura M. Ketelboeter; Sonia L. Bardy

doi:10.1371/journal.pone.0178084

Abstract

Pyomelanin is a reddish-brown pigment that provides bacteria and fungi protection from oxidative stress, and is reported to contribute to infection persistence. Production of this pigment can be inhibited by the anti-virulence agent 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). The Pseudomonas aeruginosa clinical isolate DKN343 exhibited high levels of resistance to NTBC, and the mechanism of pyomelanin production in this strain was uncharacterized. We determined that pyomelanin production in the clinical Pseudomonas aeruginosa isolate DKN343 was due to a loss of function in homogentisate 1,2-dioxygenase (HmgA). Several potential resistance mechanisms were investigated, and the MexAB-OprM efflux pump is required for resistance to NTBC. DKN343 has a frameshift mutation in NalC, which is a known indirect repressor of the mexAB-oprM operon. This frameshift mutation may contribute to the increased resistance of DKN343 to NTBC. Additional studies investigating the prevalence of resistance in pyomelanogenic microbes are necessary to determine the future applications of NTBC as an anti-virulence therapy.

Citation: Ketelboeter LM, Bardy SL (2017) Characterization of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione resistance in pyomelanogenic Pseudomonas aeruginosa DKN343. PLoS ONE 12(6): e0178084. https://doi.org/10.1371/journal.pone.0178084

Editor: Marie-Joelle Virolle, Universite Paris-Sud, FRANCE

Received: June 22, 2016; Accepted: May 7, 2017; Published: June 1, 2017

Copyright: © 2017 Ketelboeter, Bardy. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are contained within the paper, or within the Supporting Information files.

Funding: The authors received no specific funding for this work. The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Pyomelanin has several functions that are involved in helping bacteria and fungi survive in host organisms or the environment. First, pyomelanin can provide protection from oxidative stress and UV light [1–8]. Additionally, pyomelanin is involved in electron transfer, iron reduction, and iron acquisition [9–12]. There are conflicting studies on the role of pyomelanin in the infection process. The majority of reports show pyomelanin is involved in infection persistence [2, 13, 14], although in Vibrio campbellii pyomelanin production resulted in reduced virulence [15].

Pyomelanin is a negatively charged, extracellular, reddish-brown pigment that results from a defect in the tyrosine catabolism pathway. In a functional tyrosine catabolism pathway, 4-hydroxyphenylpyruvate is converted into homogentisate (HGA) by 4-hydroxyphenylpyruvate dioxygenase (Hpd) (S1 Fig). HGA is then converted into 4-maleylacetoacetate by homogentisate 1,2-dioxygenase (HmgA). Mutations or chromosomal deletions that result in loss of HmgA function lead to an accumulation of HGA [1, 2, 16, 17]. HGA is secreted from the cell via the HatABCDE ABC transporter, auto-oxidizes, and self-polymerizes to form pyomelanin [2, 18–20]. A reduction in enzyme activity in the latter part of the tyrosine catabolism pathway (HmgA, MaiA, and FahA) relative to the upper part of the pathway can also lead to pyomelanin production [21].

Many clinical and environmental bacterial species have been reported to produce pyomelanin, including Pseudomonas aeruginosa, Burkholderia cepacia complex, Vibrio cholerae, Legionella pneumophila, Shewanella algae, Bacillus anthracis, Aeromonas media, Acinetobacter baumannii, Sinorhizobium meliloti, Streptomyces coelicolor, and Alteromonas stellipolaris, [4, 7, 10, 12, 22–29]. As bacteria are increasingly developing resistance to antibiotics, investigation into new therapeutic agents is necessary [30]. Because pyomelanin has several functions that would be advantageous for bacterial survival in a host and has been reported to contribute to infection persistence, compounds that inhibit pyomelanin production are being investigated as possible therapeutic agents that could be used alone or in combination with existing antimicrobials [1, 31]. We have previously shown that the Hpd inhibiting compound 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) reduces pyomelanin production in P. aeruginosa clinical isolates and increases sensitivity to oxidative stress [1]. NTBC binds to the ferrous form of Hpd with high affinity in what has been proposed to be an irreversible reaction [32]. While this triketone was originally developed as an herbicide, it also inhibits Hpd in humans and has been employed therapeutically to treat type 1 tyrosinemia and alkaptonuria [33].

P. aeruginosa is a Gram-negative bacterium that is able to survive in a variety of different ecological environments including soil, water, plants, and animals [34]. This organism is also an opportunistic pathogen that frequently colonizes the lungs of cystic fibrosis (CF) patients, where it forms biofilms resulting in chronic infections [35]. Additionally, it causes acute infections in burn patients [36, 37]. P. aeruginosa is capable of producing a variety of virulence factors including secreted proteins, toxins, and pigments that contribute to the infection process [38].

High levels of intrinsic and acquired antimicrobial resistance are also characteristic of P. aeruginosa and occur through a variety of mechanisms. These include outer membrane impermeability, chromosomally encoded β-lactamases, increased resistance during biofilm formation, and the presence of multiple multi-drug efflux pumps [34, 39–41]. Twelve different resistance nodulation division (RND) efflux pumps have been identified in P. aeruginosa, and four of those are known to contribute to antibiotic resistance (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM) [34]. The MexAB-OprM multi-drug efflux pump is constitutively expressed, and hyperexpression of this efflux pump has been observed in multi-drug resistant clinical isolates of P. aeruginosa [40]. Hyperexpression can result from the mutagenesis of the transcriptional regulators MexR, NalC, or NalD [42–44]. Additionally the mexAB-oprM operon can be derepressed in response to environmental cues such as oxidative stress (MexR) or environmental contaminants [45]. MexAB-OprM has broad substrate specificity, which is demonstrated by its ability to extrude several different classes of antibiotics, as well as dyes, detergents, organic solvents, fatty acid synthesis inhibitors, and homoserine lactone [46]. Ultimately, the multitude of antimicrobial resistance mechanisms in P. aeruginosa make it difficult to treat infections.

In this study, we determined that the pyomelanogenic P. aeruginosa isolate DKN343 has a high level of resistance to NTBC. This strain was previously reported to produce pyomelanin through a HmgA-independent mechanism, [20] although that is not supported by our study. Sequencing of hmgA identified two point mutations, one of which alters the predicted iron cofactor binding site and results in pyomelanin production. While NTBC has previously been shown to inhibit pyomelanin production in P. aeruginosa, its effect is limited in DKN343 due to the MexAB-OprM efflux pump.

Materials and methods

Strains, plasmids, and growth conditions

A list of strains and plasmids is found in Table 1. Strains were grown on LB agar or in LB broth supplemented with gentamicin, tetracycline, or chloramphenicol where appropriate. Antibiotic concentrations were as follows: gentamicin 10 μg ml^-1 (E. coli), 50 μg ml^-1 (PAO1 hpd::tn, PAO1 hmgA::tn, and DKN343 in broth media), and 100 μg ml^-1 (DKN343 on agar); tetracycline 10 μg ml^-1 (E. coli) and 195 μg ml^-1 (DKN343); chloramphenicol 5 μg ml^-1. Strains were grown at 37°C unless otherwise indicated.

Download:

Table 1. Strains and plasmids used in this study.

https://doi.org/10.1371/journal.pone.0178084.t001

Deletion and complementation studies

In-frame deletion mutants of hpd, mexA and PA0242 in P. aeruginosa were constructed by splicing by overlap extension (SOE) PCR with PAO1 DNA as template. Primers are listed in S1 Table. The in-frame fusions were sequenced to ensure no mutations were introduced. The deletion alleles were cloned into pEX19Gm or pEX18Tc and transformed into E. coli S17-1 for mating with P. aeruginosa PAO1 hmgA::tn and DKN343, respectively. These constructs were introduced into P. aeruginosa by conjugation, and merodiploids were selected on chloramphenicol and tetracycline or gentamicin as appropriate with overnight (PAO1 hmgA::tn) or two days (DKN343) incubation. Resolution of the merodiploids was achieved through 10% sucrose counter selection. Following screening on tetracycline or gentamicin and sucrose, the deletions were confirmed by PCR or Southern blot.

Plasmids for complementation were generated by amplifying hmgA, mexA, and PA0242 from PAO1 or DKN343 using the appropriate primers (S1 Table). The resulting PCR products were sequenced and cloned into pSB109. Single point mutations of A306T and H330Y in hmgA_PAO1 were generated by site directed mutagenesis based on the QuikChange mutagenesis kit. Reversion mutations T306A and Y330H in hmgA₃₄₃ were also generated by site directed mutagenesis. PCR was performed with the appropriate mutagenic primers (S1 Table) using the appropriate pSB109-hmgA plasmid as template. The codon change was confirmed through sequencing.

In complementation studies with His-HmgA and the His-HmgA point mutants, test tubes containing LB and gentamicin were inoculated with washed overnight cultures to a concentration of OD₆₀₀ 0.05 (PAO1 hpd::tn and PAO1 hmgA::tn) or 0.1 (DKN343) and grown to OD₆₀₀ 0.2–0.3 before induction with arabinose (0–0.1%) for 1.5 h. In complementation studies with His-MexA, test tubes containing LB, gentamicin, and 0.05% arabinose were inoculated with washed overnight cultures to a concentration of OD₆₀₀ 0.05 and incubated for 24 h. Whole cell lysates were separated by SDS-PAGE. All complementation cultures were incubated for a total of 24 hours prior to being photographed.

NTBC titrations

NTBC titrations were performed as previously described [49] with gentamicin and arabinose supplementation where appropriate. Cultures were incubated for approximately 24 hours before photos were taken.

Sequencing of hmgA, hpd, and mexAB-oprM repressors from DKN343

hmgA and hpd from DKN343 were PCR amplified (for primers see S1 Table), purified, and sequenced. Three known repressors (nalC, nalD, and mexR) of mexAB-oprM were also sequenced, along with 500 bp of upstream sequence. The sequencing results were compared to P. aeruginosa PA14 to identify any mutations.

SDS-PAGE and western blots

Whole cell lysates were analyzed by SDS-PAGE, with loading based on OD₆₀₀. Protein samples were separated on 10% gels and transferred to PVDF. Membranes were probed with mouse-α-His antibody (1:3000) followed by peroxidase conjugated sheep-α-mouse antibody (1:10000). Signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate and a CCD camera with Fotodyne software.

Nitrocefin hydrolysis assay

The nitrocefin hydrolysis assay to determine outer membrane permeability was modified from previously described procedures [50, 51]. Overnight cultures were diluted 1:60 in 30 ml LB and incubated for 2 h at 37°C with shaking. Imipenem (0.25 μg ml^-1) was added for 3 h to induce β-lactamase expression before cells were harvested by centrifugation (5000 xg, 10 min). Cell concentration was normalized based on OD₆₀₀, and cell pellets were washed and concentrated in 50 mM sodium phosphate buffer (pH 7.2). Aliquots of cells were treated with 0 and 0.1 mM EDTA for 5 min. Samples were centrifuged at 15000 xg for 30 min at room temperature and β-lactamase containing supernatant was saved. β-lactamase containing supernatant was diluted in 50 mM sodium phosphate buffer (pH 7.2) and nitrocefin (Calbiochem) was added at a final concentration of 100 μM in a total volume of 1 ml. Nitrocefin hydrolysis was measured spectrophotometrically at 482 nm and β-lactamase activity (U/L) was calculated using the extinction coefficient of 17.4 mM^-1 cm^-1 for nitrocefin. The enzyme activity per OD₆₀₀ of cells was calculated and averaged over five biological replicates. Statistical analysis was performed by ANOVA, followed by Tukey HSD post-hoc analysis using R (version 3.2.4).

Photo analysis and image manipulation

All photos were taken on a Canon PowerShot A480 digital camera. Photos were cropped in Adobe Photoshop and image resolution was adjusted to 600 pixels/inch.

Results

Previous studies in our laboratory have focused on the ability of NTBC to inhibit pyomelanin production in clinical isolates of P. aeruginosa [1]. In the course of this work, we tested the efficacy of NTBC to inhibit pyomelanin production in a number of clinical and laboratory pyomelanogenic isolates of P. aeruginosa. In these studies DKN343 revealed a high level of resistance to NTBC. The ability of DKN343 to produce pyomelanin when in the presence of high concentrations of NTBC (900 μM) suggests that NTBC is unable to inhibit Hpd activity (Fig 1). DKN343 was isolated from a sputum sample (D. Newman, personal communication) and was reported to have wild type P. aeruginosa PA14 sequence in hmgA [20]. Pyomelanin production typically results from either point mutations or deletions of hmgA, or an imbalance in enzyme activity within the tyrosine catabolism pathway [2, 16, 21]. We therefore investigated the mechanism of pyomelanin production and NTBC resistance in this clinical isolate.

Download:

Fig 1. NTBC treatment reduced pigment production in pyomelanogenic P. aeruginosa.

Strains were grown in LB with the indicated concentrations of NTBC. The non-pyomelanogenic control PAO1 hpd::tn showed no pigment change in response to NTBC treatment. The pyomelanin producers PAO1 hmgA::tn and DKN343 showed dose dependent reductions in pigmentation with increasing concentrations of NTBC. PAO1 hmgA::tn was the positive control for pyomelanin production. The clinical isolate DKN343 was less affected by NTBC than the laboratory strain PAO1 hmgA::tn, as indicated by the higher concentrations of NTBC required to reduce pyomelanin production.

https://doi.org/10.1371/journal.pone.0178084.g001

Pyomelanin production in DKN343 results from a loss of function mutation in HmgA

Because NTBC binding to Hpd inhibits the synthesis of HGA [32], the ability of NTBC to partially inhibit pyomelanin production in DKN343 suggested that this pigmentation resulted from defects in the tyrosine catabolism pathway. Although hmgA_DKN343 had previously been sequenced and found to correspond to wild type PA14 hmgA [20], we identified two mutations in HmgA_DKN343 when compared to HmgA_PA14; A306T and H330Y (S2 Fig). Importantly, the H330Y mutation occurred in the HmgA iron cofactor binding site, which is expected to be required for functional HmgA. To determine if these mutations contribute to pyomelanin production through a loss of HmgA_DKN343 function, wild type hmgA_PAO1 was expressed from an arabinose inducible plasmid in DKN343 and the control PAO1 hmgA::tn (Fig 2A). Low levels of HmgA_PAO1 expression alleviated pyomelanin production in DKN343, demonstrating that pigment production does result from a defect in HmgA function. HmgA_PAO1 is 99% identical to HmgA_PA14, with only two mismatches. These mismatches occur at amino acids 226 and 303; our data shows that these mismatches between PAO1 and PA14 do not affect the function of HmgA.

Download:

Fig 2. Pyomelanin production in DKN343 results from a loss of function mutation in HmgA.

The H330Y mutation in HmgA resulted in pyomelanin production in DKN343. (A) Expression of His-HmgA from PAO1 in trans eliminated pyomelanin production in DKN343, which demonstrated that a defect in HmgA function is responsible for pyomelanin production in this strain. HmgA_H330Y did not alleviate pyomelanin production in DKN343, despite similar levels of expression as the wild type protein, indicating that the H330 residue is important for PAO1 HmgA function. HmgA_A306T was functional, as indicated by the absence of pyomelanin production in DKN343. Anti-His western blots for HmgA_PAO1 and mutated versions of His-HmgA showed increased protein expression with increasing concentrations of arabinose. PAO1 hpd::tn was the non-pyomelanogenic control, while PAO1 hmgA::tn functioned as a positive control for pyomelanin production resulting from a defect in the tyrosine catabolism pathway. (B) Overexpression of His-HmgA₃₄₃ did not alleviate pyomelanin production in DKN343. Introduction of the Y330H reversion mutation restored HmgA₃₄₃ function and alleviated pyomelanin production in DKN343. The reversion mutant HmgA_T306A was non-functional in DKN343 and pyomelanin production was retained. Anti-His western blots for HmgA₃₄₃ and both reversion mutants showed increased protein expression with increasing concentrations of arabinose.

https://doi.org/10.1371/journal.pone.0178084.g002

To ascertain whether the A306T or the H330Y mutation identified in DKN343 was responsible for the loss of function, site directed mutagenesis was used to introduce each mutation in hmgA_PAO1, and these constructs were tested for their ability to complement the DKN343 strain (Fig 2A). Expression of PAO1 HmgA_A306T eliminated pigmentation, demonstrating that this mutation does not affect enzymatic activity. PAO1 HmgA_H330Y did not alleviate pyomelanin production, which indicated the functional importance of the H330Y mutation. Reversion mutations resulting in T306A and Y330H amino acid substitutions were individually introduced into hmgA from DKN343. These constructs were expressed from an arabinose inducible plasmid in DKN343 to determine which mutation was responsible for pyomelanin production. As indicated by a loss of pyomelanin production, function was only restored to HmgA₃₄₃ when the histidine residue was present at the iron cofactor binding site (H330). Western blots revealed stable protein expression with all versions of HmgA, thereby eliminating the possibility of insufficient protein expression as the cause of pyomelanin production.

These data clearly indicate that pyomelanin production in DKN343 occurs in the conventional manner: A loss of function mutation in HmgA prevents the conversion of HGA to 4-maleylacetoacetate. The intracellular HGA is then secreted from DKN343 by the HatABCDE ABC transporter before it is auto-oxidized and self-polymerized into pyomelanin [20].

Resistance to NTBC is mediated through the MexAB-OprM multi-drug efflux pump

Studies on the NTBC-mediated inhibition of Streptomyces avermitilis 4-hydroxyphenylpyruvate dioxygenase (HppD) revealed that NTBC binds irreversibly to the ferrous metal center in the active site of the enzyme [32]. In previous studies of pyomelanogenic P. aeruginosa, NTBC treatment inhibited pyomelanin production in a dose dependent manner [1]. Early assays on DKN343 revealed 900 μM NTBC only partially inhibited pyomelanin production (Fig 1). This is a notable increase (approximately 12 fold) in the amount of NTBC required compared to the pyomelanogenic laboratory strain PAO1 hmgA::tn, and suggested this clinical isolate possessed pre-existing resistance to NTBC. Because NTBC has been proposed as a potential therapeutic agent for pyomelanogenic infections, it was necessary to ascertain the mechanism of resistance. We investigated several ways DKN343 could remain unaffected by NTBC including target alterations, sequestration of NBTC, impermeability of the outer membrane, and efflux via MexAB-OprM.

A common mechanism of antibiotic resistance in bacteria is through target alterations. We therefore investigated the possibility that the high level of resistance in DKN343 was due to the inability of NTBC to inactivate Hpd_DKN343. Analysis of the hpd_DKN343 sequence failed to identify any mutations relative to the PA14 hpd sequence, suggesting that NTBC should be able to bind and inactivate Hpd_DKN343. As the difference in NTBC sensitivity may be strain dependent (PAO1 vs PA14), we also compared the identical PA14/DKN343 sequence of Hpd with PAO1 (S3 Fig). There is one amino acid difference; an alanine is found at location 263 in PA14, and in PAO1 an aspartate is found at the similar location. It is unlikely that this difference is the cause of resistance to NTBC, as an acidic residue (glutamic acid) is found in the same location in the pig and human HppD [52]. Additionally, co-crystallization studies of NTBC with HppD (Streptomyces avermitilis) demonstrates interactions with His187, His270 and Glu349 with the phenyl ring of NTBC sandwiched between Phe336 and Phe364 [52]. These amino acids are all conserved in Hpd of P. aeruginosa PAO1 and PA14 (S3 Fig).

We also examined the P. aeruginosa genomes for proteins with homology to the active site of Hpd with the possibility that these proteins are binding and sequestering NTBC, thereby increasing the concentration needed for inhibition of Hpd and pyomelanin production. Using BlastP, we identified the hypothetical protein PA0242/PA14_03000, wherein the C-terminal region of the protein has homology to Hpd, including conservation surrounding the iron cofactor binding sites where NTBC binds (S4A Fig). Deletion of PA0242 in PAO1 hmgA::tn and PA14_03000 in DKN343 did not increase sensitivity of these strains to NTBC (S4B Fig), demonstrating that these proteins do not contribute to resistance against NTBC.

P. aeruginosa is known to have low outer membrane permeability, which can contribute to antimicrobial resistance [34]. We therefore investigated if increasing outer membrane permeability increased sensitivity to NTBC. Treatment with 0.1 mM EDTA was sufficient to increase outer membrane permeability in PAO1 hmgA::tn and DKN343 when compared to the untreated strain. Nitrocefin hydrolysis by β-lactamase released from the periplasm was used as an indicator of outer membrane permeability (S5A Fig). Treatment of pyomelanogenic strains with 0.1 mM EDTA and sub-inhibitory concentrations of NTBC (50 μM for PAO1 hmgA::tn and 900 μM for DKN343) revealed that increasing outer membrane permeability did not increase sensitivity to NTBC (S5B Fig). In both PAO1 hmgA::tn and DKN343, the relative amount of pyomelanin produced in the NTBC treated strains was very similar regardless of EDTA treatment. These results demonstrate that outer membrane impermeability does not contribute to the heightened NTBC resistance in DKN343. The non-pyomelanogenic control PAO1 hpd::tn had a similar increase in outer membrane permeability with EDTA treatment, and growth of all strains was unaffected by EDTA (data not shown).

Multi-drug efflux pumps contribute to antimicrobial resistance by extruding a broad range of compounds from the bacterial cell [41]. We therefore tested the contribution of the constitutively active multi-drug efflux pump MexAB-OprM to NTBC resistance. Increased sensitivity to NTBC was observed in the mexA deletion mutants in both PAO1 hmgA::tn and DKN343, as indicated by reduced levels of NTBC that are sufficient to inhibit pyomelanin production (50 μM for PAO1 hmgA::tn and 100 μM for DKN343). mexA encodes the membrane fusion protein of this tripartite pump. Expression of His-MexA restored resistance to NTBC in PAO1 hmgA::tn. Partial complementation was seen in DKN343ΔmexA (Fig 3A), even though expression of His-MexA was higher in DKN343ΔmexA compared to PAO1 hmgA::tnΔmexA after 24 hours (Fig 3B). This partial complementation may be the result of the epitope-tag interfering with protein interactions. Alternatively, the stoichiometry of the efflux pump components may be altered by a high level of MexA expression, thereby interfering with pump formation. Ultimately, these results indicate that the MexAB-OprM multi-drug efflux pump mediates resistance to NTBC.

Download:

Fig 3. Resistance to NTBC is mediated through the MexAB-OprM multidrug efflux pump.

(A) Deletion of mexA in PAO1 hmgA::tn and DKN343 increased sensitivity to NTBC as indicated by reduced levels of pyomelanin. Strains complemented with His-MexA showed restoration of pyomelanin production in the presence of NTBC. PAO1 hpd::tn (V) was the pyomelanin non-producer control. Cultures were grown in LB with gentamicin and 0.05% arabinose with the indicated concentrations of NTBC. (B) The relative levels of His-MexA was determined through western blotting of whole cell lysates from PAO1 hmgA::tnΔmexA and DKN343ΔmexA. MexA was expressed at higher levels in in DKN343ΔmexA compared to PAO1 hmgA::tnΔmexA. Cultures were grown for 24 hours in LB with gentamicin and 0.05% arabinose.

https://doi.org/10.1371/journal.pone.0178084.g003

As our results showed that the clinical isolate DKN343 was able to produce pyomelanin in the presence of 900 μM NTBC and the tripartite efflux pump MexAB-OprM is required for this resistance, it is tempting to speculate that overexpression of the MexAB-OprM system is responsible for the increased resistance to NTBC when compared to the laboratory strain. Expression of the mexAB-oprM operon is transcriptionally regulated by MexR, NalC and NalD [44]. Sequencing of these regulators and their corresponding upstream regions (500 bp) revealed wild type sequence for mexR and nalD, when compared to PA14. nalC_DKN343 contains a frameshift mutation that significantly alters the sequence of NalC after P149 (Fig 4.) Normally NalC regulates transcription of mexAB-oprM by repressing expression of the divergent PA3720-armR operon [43]. ArmR is an anti-repressor that functions by binding MexR and negatively impacting the ability of MexR to bind to the promoter of mexAB-oprM. The mutation in NalC of DKN343 may contribute to the increased resistance to NTBC.

Download:

Fig 4. A frameshift mutation in nalC_DKN343 significantly alters NalC sequence.

Clustal O (1.2.0) sequence alignment of P. aeruginosa PA14 NalC (PA14_16280) and DKN343 NalC amino acid sequence. nalC_DKN343 has a single base pair deletion 448 bp into the gene, resulting in a frameshift that causes a significant change in protein sequence after P149. Asterisks indicate invariant amino acids; colons indicate conservation between groups of strongly similar properties; periods indicate conservation between groups of weakly similar properties.

https://doi.org/10.1371/journal.pone.0178084.g004

Discussion

Our investigations into the possible application of NTBC as a therapeutic agent against pyomelanogenic bacteria lead us to test this triketone against the clinical P. aeruginosa isolate DKN343. Because high levels of NTBC were insufficient to inhibit pyomelanin production, we examined both the mechanism of pyomelanin production and resistance to NTBC.

DKN343 produces pyomelanin as a result of a single amino acid change in HmgA that results in a loss of function. While the identification of a loss of function mutation in HmgA that results in pyomelanin production is in contrast with the previous studies on this isolate [20], it is not entirely unexpected given a number of studies have also identified mutations in hmgA as a cause for pyomelanin production in bacteria [25, 53]. In a Burkholderia cenocepacia pyomelanogenic CF clinical isolate, a single point mutation in hmgA resulted in an amino acid change from a glycine to an arginine at residue 378, and this mutation was conserved in three of four pigmented B. cepacia complex strains [54]. This G378R mutation in B. cenocepacia was located in the iron cofactor binding region of HmgA, which could affect iron binding and subsequent enzyme function [54]. The B. cenocepacia data are in accordance with our results, in which the H330Y mutation in HmgA_DKN343 was located at one of the iron cofactor binding sites. The importance of this residue is clearly indicated by the failure of PAO1 HmgA_H330Y to alleviate pyomelanin production as well as the restoration of function through the introduction of the reversion mutation Y330H into DKN343 HmgA.

Since many bacterial and fungal pathogens produce pyomelanin, and pyomelanin has been reported to increase pathogenicity, inhibition of pyomelanin production is a potential target for a therapeutic agent. A recent study in L. pneumophila identified a compound that inhibited phenylalanine hydroxylase, which converts phenylalanine to tyrosine, and subsequently inhibited pyomelanin production [31]. This study demonstrated that targeting other enzymes in the phenylalanine/tyrosine catabolism pathway inhibits pyomelanin production in bacteria. Targeting phenylalanine hydroxylase may have limited effectiveness in the inhibition of pyomelanin production, however, as tyrosine can still be catabolized to the pyomelanin precursor HGA. Because NTBC inhibits a later step in the catabolic pathway, the ability to use both phenylalanine and tyrosine to synthesize pyomelanin is inhibited. While NTBC shows promise as a therapeutic agent against pyomelanogenic bacteria [1], its effectiveness may be limited against bacteria with a high level of pre-existing antibiotic resistance.

P. aeruginosa has high levels of intrinsic and acquired antimicrobial resistance, which can make it difficult to treat infections. Resistance to antimicrobial agents is mediated by several mechanisms, including low outer membrane permeability, several multi-drug efflux pumps, a chromosomally encoded β-lactamase, and biofilm formation [34]. This study revealed that the constitutively expressed multi-drug efflux pump MexAB-OprM was required for NTBC resistance. This was clearly indicated by the inhibition of pyomelanin production in DKN343 mexA mutants following treatment with only 100 μM NTBC as compared to the parent strain, where pyomelanin production was partially inhibited by 900 μM NTBC. MexAB-OprM contributes to intrinsic antimicrobial resistance in P. aeruginosa by extruding several classes of antibiotics, as well as dyes, detergents, organic solvents, and fatty acid synthesis inhibitors [46]. A number of recent studies on clinical and environmental P. aeruginosa isolates have emphasized the importance of the MexAB-OprM efflux system in antibiotic resistance [44, 55, 56].

The relative level of resistance to NTBC in DKN343 that could be attributed to the MexAB-OprM efflux pump was approximately 12-fold greater compared to the lab strain. Because this increased resistance could either be due to hyperexpression of mexAB-oprM or increased activity of this pump, the known transcriptional regulators mexR, nalC and nalD were sequenced. While mexR and nalD had sequences identical to PA14, nalC contained a frameshift mutation which significantly alters NalC sequence and function. It has been shown that mutations in the MexR, NalC, or NalD regulators of mexAB-oprM transcription result in hyperexpression of the operon in clinical isolates and laboratory strains of P. aeruginosa [40, 42, 43, 45, 57]. It is likely NalC_DKN343 is non-functional, which could result in increased expression of the anti-repressor ArmR. ArmR is known to interact with MexR, thereby blocking dimer formation and the ability to bind to the to the P1 region of the mexAB-oprM promoter. If ArmR is overexpressed in DKN343 due to the NalC mutation, this would lead to increased expression of mexAB-oprM [58]. We acknowledge that there may be additional, albeit minor, factors contributing to NTBC resistance in DKN343. Even with the deletion of mexA, DKN343 still has a residual amount of pyomelanin produced at 50 μM NTBC, in contrast to the lab strain PAO1 hmgA::tn, which is null for pyomelanin production at this concentration of NTBC. In summary, while NTBC is able to inhibit pyomelanin production in P. aeruginosa, the constitutively active multi-drug efflux pump MexAB-OprM may limit the effectiveness of this compound against P. aeruginosa, as evidenced by DKN343. Future studies focused on the prevalence of NTBC resistance in pyomelanogenic microbes are required to determine the potential viability of this triketone as a therapeutic targeting bacterial infections.

Supporting information

S1 Fig. Tyrosine catabolism pathway in P. aeruginosa.

Pyomelanin is produced via inactivation of HmgA, which results in secretion of HGA from the cell and subsequent auto-oxidation and self-polymerization to form pigment. NTBC binds to Hpd to prevent formation of HGA and pyomelanin production. PhhC, aromatic amino acid aminotransferase; Hpd, 4-hydroxyphenylpyruvate dioxygenase; HmgA, homogentisate 1,2-dioxygenase; MaiA, maleylacetoacetate isomerase; FahA, fumarylacetoacetase; HatABCDE, HatABCDE ABC transporter.

https://doi.org/10.1371/journal.pone.0178084.s001

(PDF)

S2 Fig. Two amino acid changes were identified in the HmgA protein sequence from the clinical isolate DKN343 compared to the HmgA sequence from P. aeruginosa PA14.

The two amino acid changes, A306T and H330Y, are highlighted in red. The H330Y change occurred in the iron cofactor binding site of HmgA. Clustal O (1.2.1) multiple sequence alignment of the HmgA protein from PA14 and DKN343 was used to identify the amino acid changes in DKN343. Asterisks indicate invariant amino acids; colons indicate conservation between groups of strongly similar properties; periods indicate conservation between groups of weakly similar properties.

https://doi.org/10.1371/journal.pone.0178084.s002

(PDF)

S3 Fig. Sequence differences in Hpd are not involved in NTBC resistance.

Clustal O (1.2.1) multiple sequence alignment of the Hpd protein from PAO1, PA14 and DKN343 was used to identify the amino acid changes in DKN343. Hpd of DKN343 and PA14 are identical, with one amino acid difference to Hpd PAO1. Asterisks indicate invariant amino acids; colons indicate conservation between groups of strongly similar properties; periods indicate conservation between groups of weakly similar properties.

https://doi.org/10.1371/journal.pone.0178084.s003

(PDF)

S4 Fig. PA0242/PA14_03000 is not involved in NTBC resistance.

(A) Clustal O (1.2.0) sequence alignment of P. aeruginosa PAO1 and PA14 Hpd, PA0242 and PA14_03000 amino acid sequence. Hpd has sequence similarity to the C-terminal portion of the hypothetical proteins PA0242 and PA14_03000. The iron cofactor binding sites for Hpd (H168, H246, and E325) are highlighted in red. Asterisks indicate invariant amino acids; colons indicate conservation between groups of strongly similar properties; periods indicate conservation between groups of weakly similar properties. (B) Deletion of PA0242/PA14_03000 had no effect on NTBC sensitivity in the pyomelanin producers hmgA::tn and DKN343, respectively. hpd::tn was the non-pyomelanogenic control, while hmgA::tn was a positive control for pyomelanin production. Cultures were grown with the indicated concentrations of NTBC.

https://doi.org/10.1371/journal.pone.0178084.s004

(PDF)

S5 Fig. An increase in outer membrane permeability does not result in increased sensitivity to NTBC in pyomelanogenic P. aeruginosa.

(A) EDTA treatment of cells increased outer membrane permeability, as measured by extracellular β-lactamase activity via nitrocefin hydrolysis. β-lactamase activity (U/L/OD₆₀₀) for each untreated parent strain (0 mM EDTA) was set to 100% and percent β-lactamase activity was calculated following 0.1 mM EDTA treatment. Five biological replicates were tested. ANOVA followed by Tukey HSD post-hoc analysis: **, p<0.01; ***, p<0.001, n.s.; not significant. (B) hmgA::tn and DKN343 were treated with 0 or 0.1 mM EDTA and sub-inhibitory concentrations (0, 50, or 900 μM) of NTBC, as indicated, for 24 hours. hmgA::tn was the positive control for pyomelanin production. Three biological replicates were tested in triplicate. A representative data set is shown here.

https://doi.org/10.1371/journal.pone.0178084.s005

(PDF)

S1 Table. Primers used in this study.

https://doi.org/10.1371/journal.pone.0178084.s006

(DOCX)

Acknowledgments

The authors thank Dianne Newman and Carrie Harwood for their generous contribution of strains. We thank Michael Mamerow, Zachery Kunkel, and Colin Opay for their contributions. University of Wisconsin Milwaukee Research Foundation holds patent no. 8,354,451; with claims broadly directed to treating or inhibiting the progression of infection of a microorganism in a patient by administering a 4-hydroxyphenylpyruvate dioxygenase-inhibiting compound such as 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). Inventors are Graham Moran and Pang He. The University of Washington P. aeruginosa transposon mutant library is supported by NIH P30 DK089507.

Author Contributions

Conceptualization: SLB.
Formal analysis: LMK.
Funding acquisition: SLB.
Investigation: LMK SLB.
Methodology: SLB.
Project administration: LMK SLB.
Resources: SLB.
Supervision: SLB.
Validation: LMK SLB.
Visualization: LMK SLB.
Writing – original draft: LMK SLB.
Writing – review & editing: LMK SLB.

References

1. Ketelboeter LM, Potharla VY, Bardy SL. NTBC treatment of the pyomelanogenic Pseudomonas aeruginosa clinical isolate PA1111 inhibits pigment production and increases sensitivity to oxidative stress. Curr Microbiol. 2014;69(3):343–8. pmid:24801336
- View Article
- PubMed/NCBI
- Google Scholar
2. Rodriguez-Rojas A, Mena A, Martin S, Borrell N, Oliver A, Blazquez J. Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection. Microbiology. 2009;155:1050–7. pmid:19332807
- View Article
- PubMed/NCBI
- Google Scholar
3. Orlandi VT, Bolognese F, Chiodaroli L, Tolker-Nielsen T, Barbieri P. Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress. Microbiology. 2015;161(12):2298–309. pmid:26419906
- View Article
- PubMed/NCBI
- Google Scholar
4. Keith KE, Killip L, He P, Moran GR, Valvano MA. Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate. J Bacteriol. 2007;189(24):9057–65. pmid:17933889
- View Article
- PubMed/NCBI
- Google Scholar
5. Schmaler-Ripcke J, Sugareva V, Gebhardt P, Winkler R, Kniemeyer O, Heinekamp T, et al. Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus. Appl Environ Microbiol. 2009;75(2):493–503. pmid:19028908
- View Article
- PubMed/NCBI
- Google Scholar
6. Almeida-Paes R, Frases S, Araújo GeS, de Oliveira MM, Gerfen GJ, Nosanchuk JD, et al. Biosynthesis and functions of a melanoid pigment produced by species of the sporothrix complex in the presence of L-tyrosine. Appl Environ Microbiol. 2012;78(24):8623–30. PMCID: PMC3502921. pmid:23042177
- View Article
- PubMed/NCBI
- Google Scholar
7. Han H, Iakovenko L, Wilson AC. Loss of homogentisate 1,2-dioxygenase activity in Bacillus anthracis results in accumulation of protective pigment. PLoS One. 2015;10(6):e0128967. PMCID: PMC4457819. pmid:26047497
- View Article
- PubMed/NCBI
- Google Scholar
8. Steinert M, Engelhard H, Flügel M, Wintermeyer E, Hacker J. The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis. Appl Environ Microbiol. 1995;61(6):2428–30. PMCID: PMC167516. pmid:7793965
- View Article
- PubMed/NCBI
- Google Scholar
9. Zheng H, Chatfield CH, Liles MR, Cianciotto NP. Secreted pyomelanin of Legionella pneumophila promotes bacterial iron uptake and growth under iron-limiting conditions. Infect Immun. 2013;81(11):4182–91. PMCID: 3811826. pmid:23980114
- View Article
- PubMed/NCBI
- Google Scholar
10. Turick CE, Tisa LS, Caccavo F. Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY. Appl Environ Microbiol. 2002;68(5):2436–44. PMCID: PMC127558. pmid:11976119
- View Article
- PubMed/NCBI
- Google Scholar
11. Turick CE, Caccavo F, Tisa LS. Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin. FEMS Microbiol Lett. 2003;220(1):99–104. pmid:12644234
- View Article
- PubMed/NCBI
- Google Scholar
12. Chatfield CH, Cianciotto NP. The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity. Infect Immun. 2007;75(8):4062–70. PMCID: PMC1951983. pmid:17548481
- View Article
- PubMed/NCBI
- Google Scholar
13. Harmer CJ, Wynn M, Pinto R, Cordwell S, Rose BR, Harbour C, et al. Homogentisate 1-2-dioxygenase downregulation in the chronic persistence of Pseudomonas aeruginosa Australian epidemic strain-1 in the CF Lung. PLoS One. 2015;10(8):e0134229. PMCID: PMC4529145. pmid:26252386
- View Article
- PubMed/NCBI
- Google Scholar
14. Valeru S, Rompikuntal P, Ishikawa T, Vaitkevicius K, Sjoling A, Dolganov N, et al. Role of melanin pigment in expression of Vibrio cholerae virulence factors. Infect Immun. 2009;77(3):935–42. PMCID: PMC2643646. pmid:19103773
- View Article
- PubMed/NCBI
- Google Scholar
15. Wang Z, Lin B, Mostaghim A, Rubin RA, Glaser ER, Mittraparp-Arthorn P, et al. Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness. Front Microbiol. 2013;4:379. PMCID: 3858670. pmid:24376440
- View Article
- PubMed/NCBI
- Google Scholar
16. Ernst RK, D'Argenio DA, Ichikawa JK, Bangera MG, Selgrade S, Burns JL, et al. Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis. Environ Microbiol. 2003;5(12):1341–9. pmid:14641578
- View Article
- PubMed/NCBI
- Google Scholar
17. Hocquet D, Petitjean M, Rohmer L, Valot B, Kulasekara H, Bedel E, et al. Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins. Environ Microbiol. 2016;18(10):3482–3493. pmid:27119970
- View Article
- PubMed/NCBI
- Google Scholar
18. Arias-Barrau E, Olivera ER, Luengo JM, Fernandez C, Galan B, Garcia JL, et al. The homogentisate pathway: a central catabolic pathway involved in the degradation of L-Phenylalanine, L-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida. J Bacteriol. 2004;186(15):5062–77. pmid:15262943
- View Article
- PubMed/NCBI
- Google Scholar
19. Turick CE, Knox AS, Becnel JM, Ekechukwu AA, Milliken CE. Properties and Function of Pyomelanin. In: Elnashar M, editor. Biopolymers In Tech2010. p. 449–72.
20. Hunter RC, Newman DK. A putative ABC transporter, HatABCDE, is among molecular determinants of pyomelanin production in Pseudomonas aeruginosa. J Bacteriol. 2010;192(22):5962–71. pmid:20870774
- View Article
- PubMed/NCBI
- Google Scholar
21. Sanchez-Amat A, Ruzafa C, Solano F. Comparative tyrosine degradation in Vibrio cholerae strains. The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell. Comp Biochem Physiol B Biochem Mol Biol. 1998;119(3):557–62. pmid:9734339
- View Article
- PubMed/NCBI
- Google Scholar
22. Yabuuchi E, Ohyama A. Characterization of "Pyomelanin"-Producing Strains of Pseudomonas aeruginosa. Int J Syst Bacteriol. 1972;22(2):53–64.
- View Article
- Google Scholar
23. Zughaier SM, Ryley HC, Jackson SK. A melanin pigment purified from an epidemic strain of Burkholderia cepacia attenuates monocyte respiratory burst activity by scavenging superoxide anion. Infect Immun. 1999;67(2):908–13. pmid:9916107
- View Article
- PubMed/NCBI
- Google Scholar
24. Kotob SI, Coon SL, Quintero EJ, Weiner RM. Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Appl Environ Microbiol. 1995;61(4):1620–2. pmid:7747976
- View Article
- PubMed/NCBI
- Google Scholar
25. Wang H, Qiao Y, Chai B, Qiu C, Chen X. Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS. PLoS One. 2015;10(3):e0120923. PMCID: PMC4368426. pmid:25793756
- View Article
- PubMed/NCBI
- Google Scholar
26. Coelho-Souza T, Martins N, Maia F, Frases S, Bonelli RR, Riley LW, et al. Pyomelanin production: a rare phenotype in Acinetobacter baumannii. J Med Microbiol. 2014;63(Pt 1):152–4. PMCID: PMC3870426. pmid:24149622
- View Article
- PubMed/NCBI
- Google Scholar
27. Milcamps A, de Bruijn FJ. Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine. Microbiology. 1999;145(Pt 4):935–47.
- View Article
- Google Scholar
28. Yang H, Wang L, Xie Z, Tian Y, Liu G, Tan H. The tyrosine degradation gene hppD is transcriptionally activated by HpdA and repressed by HpdR in Streptomyces coelicolor, while hpdA is negatively autoregulated and repressed by HpdR. Mol Microbiol. 2007;65(4):1064–77. pmid:17640269
- View Article
- PubMed/NCBI
- Google Scholar
29. Zhu S, Lu Y, Xu X, Chen J, Yang J, Ma X. Isolation and identification of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856. Folia Microbiol (Praha). 2015;60(4):309–16.
- View Article
- Google Scholar
30. AR H, J M, DT M. Beyond antibiotics: New therapeutic approaches for bacterial infections. Clinical Infectious Disease. 2016.
- View Article
- Google Scholar
31. Aubi O, Flydal MI, Zheng H, Skjærven L, Rekand I, Leiros HK, et al. Discovery of a specific inhibitor of pyomelanin synthesis in Legionella pneumophila. J Med Chem. 2015;58(21):8402–12. pmid:26458252
- View Article
- PubMed/NCBI
- Google Scholar
32. Kavana M, Moran GR. Interaction of (4-Hydroxyphenyl)pyruvate dioxygenase with the specific inhibitor 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione. Biochemistry. 2003;42(34):10238–45. pmid:12939152
- View Article
- PubMed/NCBI
- Google Scholar
33. Holme E, Lindstedt S. Tyrosinaemia type I and NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione). J Inherit Metab Dis. 1998;21(5):507–17. pmid:9728331
- View Article
- PubMed/NCBI
- Google Scholar
34. El Zowalaty ME, Al Thani AA, Webster TJ, El Zowalaty AE, Schweizer HP, Nasrallah GK, et al. Pseudomonas aeruginosa: arsenal of resistance mechanisms, decades of changing resistance profiles, and future antimicrobial therapies. Future Microbiol. 2015;10(10):1683–706. pmid:26439366
- View Article
- PubMed/NCBI
- Google Scholar
35. Hassett DJ, Sutton MD, Schurr MJ, Herr AB, Caldwell CC, Matu JO. Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways. Trends Microbiol. 2009;17(3):130–8. pmid:19231190
- View Article
- PubMed/NCBI
- Google Scholar
36. Azimi L, Motevallian A, Namvar AE, Asghari B, Lari AR. Nosocomial infections in burned patients in Motahari Hospital, Tehran, Iran. Dermatol Res Pract. 2011;2011:436952. pmid:22203838
- View Article
- PubMed/NCBI
- Google Scholar
37. Vahdani M, Azimi L, Asghari B, Bazmi F, Rastegar Lari A. Phenotypic screening of extended-spectrum beta-lactamase and metallo-beta-lactamase in multidrug-resistant Pseudomonas aeruginosa from infected burns. Ann Burns Fire Disasters. 2012;25(2):78–81. pmid:23233825
- View Article
- PubMed/NCBI
- Google Scholar
38. Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect. 2006;36(2):78–91. pmid:16427231
- View Article
- PubMed/NCBI
- Google Scholar
39. Delcour AH. Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta. 2009;1794(5):808–16. PMCID: PMC2696358. pmid:19100346
- View Article
- PubMed/NCBI
- Google Scholar
40. De Kievit TR, Parkins MD, Gillis RJ, Srikumar R, Ceri H, Poole K, et al. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2001;45(6):1761–70. PMCID: PMC90543. pmid:11353623
- View Article
- PubMed/NCBI
- Google Scholar
41. Dreier J, Ruggerone P. Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa. Front Microbiol. 2015;6:660. PMCID: PMC4495556. pmid:26217310
- View Article
- PubMed/NCBI
- Google Scholar
42. Sobel ML, Hocquet D, Cao L, Plesiat P, Poole K. Mutations in PA3574 (nalD) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2005;49(5):1782–6. PMCID: PMC1087681. pmid:15855496
- View Article
- PubMed/NCBI
- Google Scholar
43. Cao L, Srikumar R, Poole K. MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719. Mol Microbiol. 2004;53(5):1423–36. pmid:15387820
- View Article
- PubMed/NCBI
- Google Scholar
44. Braz VS, Furlan JP, Fernandes AF, Stehling EG. Mutations in NalC induce MexAB-OprM overexpression resulting in high level of aztreonam resistance in environmental isolates of Pseudomonas aeruginosa. FEMS Microbiol Lett. 2016;363(16). Epub 2016 Jul 12.
- View Article
- Google Scholar
45. Starr LM, Fruci M, Poole K. Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR. PLoS One. 2012;7(2):e32684. PMCID: PMC3290565. pmid:22393435
- View Article
- PubMed/NCBI
- Google Scholar
46. Chen W, Wang D, Zhou W, Sang H, Liu X, Ge Z, et al. Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa. Mol Microbiol. 2016.
- View Article
- Google Scholar
47. Simon R, Priefer U, Puhler A. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram negative bacteria. Biotechnology. 1983;1(9):784–91.
- View Article
- Google Scholar
48. Hoang T, Karkhoff-Schweizer R, Kutchma A, Schweizer H. A broad-host-range Flp-FRT recombination system for site-specific excision of the chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene. 1998;212(1):77–86. pmid:9661666
- View Article
- PubMed/NCBI
- Google Scholar
49. Ketelboeter LM, Bardy SL. Methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress. J Vis Exp. 2015;102:e53105.
- View Article
- Google Scholar
50. Li XZ, Zhang L, Srikumar R, Poole K. Beta-lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1998;42(2):399–403. PMCID: PMC105421. pmid:9527793
- View Article
- PubMed/NCBI
- Google Scholar
51. Li XZ, Zhang L, Poole K. Interplay between the MexA-MexB-OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa. J Antimicrob Chemother. 2000;45(4):433–6. pmid:10747818
- View Article
- PubMed/NCBI
- Google Scholar
52. Brownlee J, Johnson-Winters K, Harrison D, Moran GR. Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide, NTBC. Biochemistry. 2004;43(21):6370–7. pmid:15157070
- View Article
- PubMed/NCBI
- Google Scholar
53. Wang R, Wang H, Zhou H, Wang Y, Yue J, Diao B, et al. Characters of homogentisate oxygenase gene mutation and high clonality of the natural pigment-producing Vibrio cholerae strains. BMC Microbiol. 2011;11:109. PMCID: PMC3114702. pmid:21592381
- View Article
- PubMed/NCBI
- Google Scholar
54. Gonyar LA, Fankhauser SC, Goldberg JB. Single amino acid substitution in homogentisate 1,2-dioxygenase is responsible for pigmentation in a subset of Burkholderia cepacia complex isolates. Environ Microbiol Rep. 2015;7(2):180–7. PMCID: PMC4560265. pmid:25294803
- View Article
- PubMed/NCBI
- Google Scholar
55. Aghazadeh M, Hojabri Z, Mahdian R, Nahaei M, Rahmati M, Hojabri T, et al. Role of efflux pumps: MexAB-OprM and MexXY(-OprA), AmpC cephalosporinase and OprD porin in non-metallo-β-lactamase producing Pseudomonas aeruginosa isolated from cystic fibrosis and burn patients. Infect Genet Evol. 2014 24:187–92. pmid:24694825
- View Article
- PubMed/NCBI
- Google Scholar
56. Castanheira M, Mills J, Farrell D, Jones R. Mutation driven β-lactam resistance mechanisms among contemporary ceftazidime-nonsusceptible Pseudomonas aeruginosa isolates from U.S. hospitals. Antimicrob Agents Chemother. 2014;58(11):6844–50. pmid:25182652
- View Article
- PubMed/NCBI
- Google Scholar
57. Breidenstein EB, de la Fuente-Núñez C, Hancock RE. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 2011;19(8):419–26. pmid:21664819
- View Article
- PubMed/NCBI
- Google Scholar
58. Evans K, Adewoye L, Poole K. MexR repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa: identification of MexR binding sites in the mexA-mexR intergenic region. J Bacteriol. 2001;183:807–12. pmid:11208776
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Ketelboeter LM, Potharla VY, Bardy SL. NTBC treatment of the pyomelanogenic Pseudomonas aeruginosa clinical isolate PA1111 inhibits pigment production and increases sensitivity to oxidative stress. Curr Microbiol. 2014;69(3):343–8. pmid:24801336
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Rodriguez-Rojas A, Mena A, Martin S, Borrell N, Oliver A, Blazquez J. Inactivation of the hmgA gene of Pseudomonas aeruginosa leads to pyomelanin hyperproduction, stress resistance and increased persistence in chronic lung infection. Microbiology. 2009;155:1050–7. pmid:19332807
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Orlandi VT, Bolognese F, Chiodaroli L, Tolker-Nielsen T, Barbieri P. Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress. Microbiology. 2015;161(12):2298–309. pmid:26419906
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Keith KE, Killip L, He P, Moran GR, Valvano MA. Burkholderia cenocepacia C5424 produces a pigment with antioxidant properties using a homogentisate intermediate. J Bacteriol. 2007;189(24):9057–65. pmid:17933889
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Schmaler-Ripcke J, Sugareva V, Gebhardt P, Winkler R, Kniemeyer O, Heinekamp T, et al. Production of pyomelanin, a second type of melanin, via the tyrosine degradation pathway in Aspergillus fumigatus. Appl Environ Microbiol. 2009;75(2):493–503. pmid:19028908
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Almeida-Paes R, Frases S, Araújo GeS, de Oliveira MM, Gerfen GJ, Nosanchuk JD, et al. Biosynthesis and functions of a melanoid pigment produced by species of the sporothrix complex in the presence of L-tyrosine. Appl Environ Microbiol. 2012;78(24):8623–30. PMCID: PMC3502921. pmid:23042177
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Han H, Iakovenko L, Wilson AC. Loss of homogentisate 1,2-dioxygenase activity in Bacillus anthracis results in accumulation of protective pigment. PLoS One. 2015;10(6):e0128967. PMCID: PMC4457819. pmid:26047497
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Steinert M, Engelhard H, Flügel M, Wintermeyer E, Hacker J. The Lly protein protects Legionella pneumophila from light but does not directly influence its intracellular survival in Hartmannella vermiformis. Appl Environ Microbiol. 1995;61(6):2428–30. PMCID: PMC167516. pmid:7793965
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Zheng H, Chatfield CH, Liles MR, Cianciotto NP. Secreted pyomelanin of Legionella pneumophila promotes bacterial iron uptake and growth under iron-limiting conditions. Infect Immun. 2013;81(11):4182–91. PMCID: 3811826. pmid:23980114
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Turick CE, Tisa LS, Caccavo F. Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY. Appl Environ Microbiol. 2002;68(5):2436–44. PMCID: PMC127558. pmid:11976119
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Turick CE, Caccavo F, Tisa LS. Electron transfer from Shewanella algae BrY to hydrous ferric oxide is mediated by cell-associated melanin. FEMS Microbiol Lett. 2003;220(1):99–104. pmid:12644234
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Chatfield CH, Cianciotto NP. The secreted pyomelanin pigment of Legionella pneumophila confers ferric reductase activity. Infect Immun. 2007;75(8):4062–70. PMCID: PMC1951983. pmid:17548481
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Harmer CJ, Wynn M, Pinto R, Cordwell S, Rose BR, Harbour C, et al. Homogentisate 1-2-dioxygenase downregulation in the chronic persistence of Pseudomonas aeruginosa Australian epidemic strain-1 in the CF Lung. PLoS One. 2015;10(8):e0134229. PMCID: PMC4529145. pmid:26252386
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Valeru S, Rompikuntal P, Ishikawa T, Vaitkevicius K, Sjoling A, Dolganov N, et al. Role of melanin pigment in expression of Vibrio cholerae virulence factors. Infect Immun. 2009;77(3):935–42. PMCID: PMC2643646. pmid:19103773
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Wang Z, Lin B, Mostaghim A, Rubin RA, Glaser ER, Mittraparp-Arthorn P, et al. Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness. Front Microbiol. 2013;4:379. PMCID: 3858670. pmid:24376440
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Ernst RK, D'Argenio DA, Ichikawa JK, Bangera MG, Selgrade S, Burns JL, et al. Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis. Environ Microbiol. 2003;5(12):1341–9. pmid:14641578
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Hocquet D, Petitjean M, Rohmer L, Valot B, Kulasekara H, Bedel E, et al. Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins. Environ Microbiol. 2016;18(10):3482–3493. pmid:27119970
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Arias-Barrau E, Olivera ER, Luengo JM, Fernandez C, Galan B, Garcia JL, et al. The homogentisate pathway: a central catabolic pathway involved in the degradation of L-Phenylalanine, L-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida. J Bacteriol. 2004;186(15):5062–77. pmid:15262943
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Turick CE, Knox AS, Becnel JM, Ekechukwu AA, Milliken CE. Properties and Function of Pyomelanin. In: Elnashar M, editor. Biopolymers In Tech2010. p. 449–72.

[ref20] 20. Hunter RC, Newman DK. A putative ABC transporter, HatABCDE, is among molecular determinants of pyomelanin production in Pseudomonas aeruginosa. J Bacteriol. 2010;192(22):5962–71. pmid:20870774
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref21] 21. Sanchez-Amat A, Ruzafa C, Solano F. Comparative tyrosine degradation in Vibrio cholerae strains. The strain ATCC 14035 as a prokaryotic melanogenic model of homogentisate-releasing cell. Comp Biochem Physiol B Biochem Mol Biol. 1998;119(3):557–62. pmid:9734339
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref22] 22. Yabuuchi E, Ohyama A. Characterization of "Pyomelanin"-Producing Strains of Pseudomonas aeruginosa. Int J Syst Bacteriol. 1972;22(2):53–64.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref23] 23. Zughaier SM, Ryley HC, Jackson SK. A melanin pigment purified from an epidemic strain of Burkholderia cepacia attenuates monocyte respiratory burst activity by scavenging superoxide anion. Infect Immun. 1999;67(2):908–13. pmid:9916107
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref24] 24. Kotob SI, Coon SL, Quintero EJ, Weiner RM. Homogentisic acid is the primary precursor of melanin synthesis in Vibrio cholerae, a Hyphomonas strain, and Shewanella colwelliana. Appl Environ Microbiol. 1995;61(4):1620–2. pmid:7747976
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref25] 25. Wang H, Qiao Y, Chai B, Qiu C, Chen X. Identification and molecular characterization of the homogentisate pathway responsible for pyomelanin production, the major melanin constituents in Aeromonas media WS. PLoS One. 2015;10(3):e0120923. PMCID: PMC4368426. pmid:25793756
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref26] 26. Coelho-Souza T, Martins N, Maia F, Frases S, Bonelli RR, Riley LW, et al. Pyomelanin production: a rare phenotype in Acinetobacter baumannii. J Med Microbiol. 2014;63(Pt 1):152–4. PMCID: PMC3870426. pmid:24149622
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref27] 27. Milcamps A, de Bruijn FJ. Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine. Microbiology. 1999;145(Pt 4):935–47.
View Article
Google Scholar

[102] View Article

[103] Google Scholar

[ref28] 28. Yang H, Wang L, Xie Z, Tian Y, Liu G, Tan H. The tyrosine degradation gene hppD is transcriptionally activated by HpdA and repressed by HpdR in Streptomyces coelicolor, while hpdA is negatively autoregulated and repressed by HpdR. Mol Microbiol. 2007;65(4):1064–77. pmid:17640269
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref29] 29. Zhu S, Lu Y, Xu X, Chen J, Yang J, Ma X. Isolation and identification of a gene encoding 4-hydroxyphenylpyruvate dioxygenase from the red-brown pigment-producing bacterium Alteromonas stellipolaris LMG 21856. Folia Microbiol (Praha). 2015;60(4):309–16.
View Article
Google Scholar

[109] View Article

[110] Google Scholar

[ref30] 30. AR H, J M, DT M. Beyond antibiotics: New therapeutic approaches for bacterial infections. Clinical Infectious Disease. 2016.
View Article
Google Scholar

[112] View Article

[113] Google Scholar

[ref31] 31. Aubi O, Flydal MI, Zheng H, Skjærven L, Rekand I, Leiros HK, et al. Discovery of a specific inhibitor of pyomelanin synthesis in Legionella pneumophila. J Med Chem. 2015;58(21):8402–12. pmid:26458252
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref32] 32. Kavana M, Moran GR. Interaction of (4-Hydroxyphenyl)pyruvate dioxygenase with the specific inhibitor 2-[2-Nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione. Biochemistry. 2003;42(34):10238–45. pmid:12939152
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref33] 33. Holme E, Lindstedt S. Tyrosinaemia type I and NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione). J Inherit Metab Dis. 1998;21(5):507–17. pmid:9728331
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref34] 34. El Zowalaty ME, Al Thani AA, Webster TJ, El Zowalaty AE, Schweizer HP, Nasrallah GK, et al. Pseudomonas aeruginosa: arsenal of resistance mechanisms, decades of changing resistance profiles, and future antimicrobial therapies. Future Microbiol. 2015;10(10):1683–706. pmid:26439366
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref35] 35. Hassett DJ, Sutton MD, Schurr MJ, Herr AB, Caldwell CC, Matu JO. Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways. Trends Microbiol. 2009;17(3):130–8. pmid:19231190
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref36] 36. Azimi L, Motevallian A, Namvar AE, Asghari B, Lari AR. Nosocomial infections in burned patients in Motahari Hospital, Tehran, Iran. Dermatol Res Pract. 2011;2011:436952. pmid:22203838
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref37] 37. Vahdani M, Azimi L, Asghari B, Bazmi F, Rastegar Lari A. Phenotypic screening of extended-spectrum beta-lactamase and metallo-beta-lactamase in multidrug-resistant Pseudomonas aeruginosa from infected burns. Ann Burns Fire Disasters. 2012;25(2):78–81. pmid:23233825
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref38] 38. Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect. 2006;36(2):78–91. pmid:16427231
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref39] 39. Delcour AH. Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta. 2009;1794(5):808–16. PMCID: PMC2696358. pmid:19100346
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref40] 40. De Kievit TR, Parkins MD, Gillis RJ, Srikumar R, Ceri H, Poole K, et al. Multidrug efflux pumps: expression patterns and contribution to antibiotic resistance in Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother. 2001;45(6):1761–70. PMCID: PMC90543. pmid:11353623
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref41] 41. Dreier J, Ruggerone P. Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa. Front Microbiol. 2015;6:660. PMCID: PMC4495556. pmid:26217310
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref42] 42. Sobel ML, Hocquet D, Cao L, Plesiat P, Poole K. Mutations in PA3574 (nalD) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2005;49(5):1782–6. PMCID: PMC1087681. pmid:15855496
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref43] 43. Cao L, Srikumar R, Poole K. MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719. Mol Microbiol. 2004;53(5):1423–36. pmid:15387820
View Article
PubMed/NCBI
Google Scholar

[163] View Article

[164] PubMed/NCBI

[165] Google Scholar

[ref44] 44. Braz VS, Furlan JP, Fernandes AF, Stehling EG. Mutations in NalC induce MexAB-OprM overexpression resulting in high level of aztreonam resistance in environmental isolates of Pseudomonas aeruginosa. FEMS Microbiol Lett. 2016;363(16). Epub 2016 Jul 12.
View Article
Google Scholar

[167] View Article

[168] Google Scholar

[ref45] 45. Starr LM, Fruci M, Poole K. Pentachlorophenol induction of the Pseudomonas aeruginosa mexAB-oprM efflux operon: involvement of repressors NalC and MexR and the antirepressor ArmR. PLoS One. 2012;7(2):e32684. PMCID: PMC3290565. pmid:22393435
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref46] 46. Chen W, Wang D, Zhou W, Sang H, Liu X, Ge Z, et al. Novobiocin binding to NalD induces the expression of the MexAB-OprM pump in Pseudomonas aeruginosa. Mol Microbiol. 2016.
View Article
Google Scholar

[174] View Article

[175] Google Scholar

[ref47] 47. Simon R, Priefer U, Puhler A. A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in Gram negative bacteria. Biotechnology. 1983;1(9):784–91.
View Article
Google Scholar

[177] View Article

[178] Google Scholar

[ref48] 48. Hoang T, Karkhoff-Schweizer R, Kutchma A, Schweizer H. A broad-host-range Flp-FRT recombination system for site-specific excision of the chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene. 1998;212(1):77–86. pmid:9661666
View Article
PubMed/NCBI
Google Scholar

[180] View Article

[181] PubMed/NCBI

[182] Google Scholar

[ref49] 49. Ketelboeter LM, Bardy SL. Methods to inhibit bacterial pyomelanin production and determine the corresponding increase in sensitivity to oxidative stress. J Vis Exp. 2015;102:e53105.
View Article
Google Scholar

[184] View Article

[185] Google Scholar

[ref50] 50. Li XZ, Zhang L, Srikumar R, Poole K. Beta-lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1998;42(2):399–403. PMCID: PMC105421. pmid:9527793
View Article
PubMed/NCBI
Google Scholar

[187] View Article

[188] PubMed/NCBI

[189] Google Scholar

[ref51] 51. Li XZ, Zhang L, Poole K. Interplay between the MexA-MexB-OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa. J Antimicrob Chemother. 2000;45(4):433–6. pmid:10747818
View Article
PubMed/NCBI
Google Scholar

[191] View Article

[192] PubMed/NCBI

[193] Google Scholar

[ref52] 52. Brownlee J, Johnson-Winters K, Harrison D, Moran GR. Structure of the ferrous form of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis in complex with the therapeutic herbicide, NTBC. Biochemistry. 2004;43(21):6370–7. pmid:15157070
View Article
PubMed/NCBI
Google Scholar

[195] View Article

[196] PubMed/NCBI

[197] Google Scholar

[ref53] 53. Wang R, Wang H, Zhou H, Wang Y, Yue J, Diao B, et al. Characters of homogentisate oxygenase gene mutation and high clonality of the natural pigment-producing Vibrio cholerae strains. BMC Microbiol. 2011;11:109. PMCID: PMC3114702. pmid:21592381
View Article
PubMed/NCBI
Google Scholar

[199] View Article

[200] PubMed/NCBI

[201] Google Scholar

[ref54] 54. Gonyar LA, Fankhauser SC, Goldberg JB. Single amino acid substitution in homogentisate 1,2-dioxygenase is responsible for pigmentation in a subset of Burkholderia cepacia complex isolates. Environ Microbiol Rep. 2015;7(2):180–7. PMCID: PMC4560265. pmid:25294803
View Article
PubMed/NCBI
Google Scholar

[203] View Article

[204] PubMed/NCBI

[205] Google Scholar

[ref55] 55. Aghazadeh M, Hojabri Z, Mahdian R, Nahaei M, Rahmati M, Hojabri T, et al. Role of efflux pumps: MexAB-OprM and MexXY(-OprA), AmpC cephalosporinase and OprD porin in non-metallo-β-lactamase producing Pseudomonas aeruginosa isolated from cystic fibrosis and burn patients. Infect Genet Evol. 2014 24:187–92. pmid:24694825
View Article
PubMed/NCBI
Google Scholar

[207] View Article

[208] PubMed/NCBI

[209] Google Scholar

[ref56] 56. Castanheira M, Mills J, Farrell D, Jones R. Mutation driven β-lactam resistance mechanisms among contemporary ceftazidime-nonsusceptible Pseudomonas aeruginosa isolates from U.S. hospitals. Antimicrob Agents Chemother. 2014;58(11):6844–50. pmid:25182652
View Article
PubMed/NCBI
Google Scholar

[211] View Article

[212] PubMed/NCBI

[213] Google Scholar

[ref57] 57. Breidenstein EB, de la Fuente-Núñez C, Hancock RE. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 2011;19(8):419–26. pmid:21664819
View Article
PubMed/NCBI
Google Scholar

[215] View Article

[216] PubMed/NCBI

[217] Google Scholar

[ref58] 58. Evans K, Adewoye L, Poole K. MexR repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa: identification of MexR binding sites in the mexA-mexR intergenic region. J Bacteriol. 2001;183:807–12. pmid:11208776
View Article
PubMed/NCBI
Google Scholar

[219] View Article

[220] PubMed/NCBI

[221] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Strains, plasmids, and growth conditions

Deletion and complementation studies

NTBC titrations

Sequencing of hmgA, hpd, and mexAB-oprM repressors from DKN343

SDS-PAGE and western blots

Nitrocefin hydrolysis assay

Photo analysis and image manipulation

Results

Pyomelanin production in DKN343 results from a loss of function mutation in HmgA

Resistance to NTBC is mediated through the MexAB-OprM multi-drug efflux pump

Discussion

Supporting information

S1 Fig. Tyrosine catabolism pathway in P. aeruginosa.

S2 Fig. Two amino acid changes were identified in the HmgA protein sequence from the clinical isolate DKN343 compared to the HmgA sequence from P. aeruginosa PA14.

S3 Fig. Sequence differences in Hpd are not involved in NTBC resistance.

S4 Fig. PA0242/PA14_03000 is not involved in NTBC resistance.

S5 Fig. An increase in outer membrane permeability does not result in increased sensitivity to NTBC in pyomelanogenic P. aeruginosa.

S1 Table. Primers used in this study.

Acknowledgments

Author Contributions

References