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Oceanic facilities and equipment corrosion present considerable economic and safety concerns, predominantly due to microbial corrosion. Early detection of corrosive microbes is pivotal for effective monitoring and prevention. Yet, traditional detection methods often lack specificity, require extensive processing time, and yield inaccurate results. Hence, the need for an efficient real-time corrosive microbe monitoring technology is evident. Pseudomonas aeruginosa, a widely distributed microorganism in aquatic environments, utilizes its production of quinone-like compounds, specifically pyocyanin (PYO), to corrode metals. Here, we report a novel fiber optic surface plasmon resonance (SPR) sensor modified by the C-terminal of BrlR protein (BrlR-C), which is a specific receptor of PYO molecule, to detect P. aeruginosa in aquatic environments. The results showed that the sensor had a good ability to recognize PYO in the concentration range of 0-1 μg/mL, and showed excellent sensing performance in real-time monitoring the growth status of P. aeruginosa. With a strong selectivity of PYO, the sensor could clearly detect P. aeruginosa against other bacteria in seawater environment, and exhibited excellent anti-interference ability against variations in pH, temperature and pressure and other interfering substances. This study provides a useful tool for monitoring corrosive P. aeruginosa biofilm in aquatic environments, which is a first of its kind example that serves as a laboratory model for the application of fiber optic technology in real-world scenarios to monitoring biofilms in microbial corrosion and biofouling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Combating Pseudomonas aeruginosa infection is challenging. It secretes pyocyanin (PCN) pigment that contributes to its virulence. Neutralizing PCN via reaction with thiol-containing compounds may represent a potential therapeutic option. This study investigates the neutralization reaction between PCN and N-acetyl cysteine (NAC) for bacterial inhibition and explores its mechanism of action. The neutralization adduct (PCN-NAC) was synthesized by reacting the purified PCN and NAC. The adduct was analyzed and its structure was elucidated. LC-MS/MS method was developed for the determination of PCN-NAC in P. aeruginosa cultures post-treatment with NAC (0-5 mg/mL). The corresponding anti-bacterial potential was estimated and compared to nanoparticles (NPs) alone and under stress conditions. In silico studies were performed to support explaining the mechanism of action. Results revealed that PCN-NAC was exclusively detected in NAC-treated cultures in a concentration-dependent manner. PCN-NAC concentration (230-915 µg/mL) was directly proportional to the reduction in the bacterial viable count (28.3% ± 7.1-87.5% ± 5.9) and outperformed all tested NPs, where chitosan NPs induced 56.9% ± 7.9 inhibition, followed by zinc NPs (49.4% ± 0.9) and gold NPs (17.8% ± 7.5) even post-exposure to different stress conditions. A concomitant reduction in PCN concentration was detected. In silico studies revealed possible interactions between key bacterial proteins and PCN-NAC rather than the NAC itself. These results pose NAC as a potential choice for the management of P. aeruginosa infection, where it neutralizes PCN via the formation of PCN-NAC adduct., (© 2024. The Author(s), under exclusive licence to The Japan Society for Analytical Chemistry.)

Repair of epithelial defect is complicated by infection and related metabolites. Pyocyanin (PYO) is one such metabolite that is secreted during Pseudomonas aeruginosa infection. Keratinocyte (KC) migration is required for the closure of skin epithelial defects. This work sought to understand PYO-KC interaction and its significance in tissue repair. Stable Isotope Labeling by Amino acids in Cell culture proteomics identified mitochondrial dysfunction as the top pathway responsive to PYO exposure in human KCs. Consistently, functional studies showed mitochondrial stress, depletion of reducing equivalents, and adenosine triphosphate. Strikingly, despite all stated earlier, PYO markedly accelerated KC migration. Investigation of underlying mechanisms revealed, to our knowledge, a previously unreported function of keratin 6A in KCs. Keratin 6A was PYO inducible and accelerated closure of epithelial defect. Acceleration of closure was associated with poor quality healing, including compromised expression of apical junction proteins. This work recognizes keratin 6A for its role in enhancing KC migration under conditions of threat posed by PYO. Qualitatively deficient junctional proteins under conditions of defensive acceleration of KC migration explain why an infected wound close with deficient skin barrier function as previously reported., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The electrochemical biosensing strategy for pyocyanin (PYO), a virulent quorum-sensing molecule responsible for Pseudomonas aeruginosa infections, was developed by mimicking its extracellular DNA interaction. Calf thymus DNA (ct-DNA) functionalized amine-containing carbon quantum dots (CQDs) were used as a biomimetic receptor for electrochemical sensing of PYO as low as 37 nM in real urine sample. The ct-DNA-based biosensor enabled the selective measurement of PYO in the presence of other interfering species. Calibration and validation of the PYO sensor platform were demonstrated in buffer solution (0-100 μM), microbial culture media (0-100 μM), artificial urine (0-400 μM), and real urine sample (0-250 μM). The sensor capability was successfully implemented for point-of-care (POC) detection of PYO release from Pseudomonas aeruginosa strains during lag and stationary phases. Cross-reactivity of the sensing platform was also tested in other bacterial species such as Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Shigella dysenteriae, Staphylococcus aureus, and Streptococcus pneumoniae. Potential clinical implementation of the ct-DNA-based sensor was manifested in detecting the PYO in P. aeruginosa cultured baby diaper and sanitary napkin. Our results highlight that the newly developed ct-DNA-based sensing platform can be used as a potential candidate for real-time POC diagnosis of Pseudomonas aeruginosa infection in clinical samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The antibiotic crisis is associated with the appearance of multidrug resistant (MDR) pathogens, which has caused severe bacterial infections and imposed a huge burden on modern society. Therefore, there is an urgent need to develop new antibacterial drugs with novel mechanism of action. Here we designed and synthesized three series of benzoxazolone, oxazolopyridinone and 3-(2-hydroxyphenyl)hydantoin derivatives and evaluated their activity as novel quorum sensing (QS) inhibitors. We found that benzoxazolone and oxazolopyridinone derivatives had promising QS inhibitory activity in the minimum inhibitory concentration, pyocyanin and rhamnolipid inhibition assays. In particular, A10 and B20 at 256 μg/mL not only suppressed pyocyanin production regulated by QS in P. aeruginosa PAO1 by 36.55% and 46.90%, respectively, but also showed the strongest rhamnolipid inhibitory activity with the IC 50 values of 66.35 and 56.75 µg/mL, respectively. Further studies demonstrated that B20 at 64 μg/mL inhibited biofilm formation in P. aeruginosa PAO1 by 40%, and weakened its swarming motility. More importantly, the bacterial mortality of B20 combined with ciprofloxacin and clarithromycin against P. aeruginosa were 48.27% and 49.79%, respectively, while ciprofloxacin and clarithromycin had only 16.99% and 29.11% of bacterial mortality against P. aeruginosa when used alone. Mechanistic studies indicated that B20 directly inhibited the QS pathway based on the GFP reporter strain assay. Overall, this compound with oxazolopyridinone core could serve as an antibacterial lead of QS inhibitor for further evaluation of its drug-likeness., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Quorum-sensing (QS) systems of Pseudomonas aeruginosa  are involved in the control of biofilm formation and virulence factor production. The current study evaluated the ability of halogenated dihydropyrrol-2-ones (DHP) (Br (4a), Cl (4b), and F (4c)) and a non-halogenated version (4d) to inhibit the QS receptor proteins LasR and PqsR. The DHP molecules exhibited concentration-dependent inhibition of LasR and PqsR receptor proteins. For LasR, all compounds showed similar inhibition levels. However, compound 4a (Br) showed the highest decrease (two-fold) for PqsR, even at the lowest concentration (12.5 µg/mL). Inhibition of QS decreased pyocyanin production amongst P . aeruginosa PAO1, MH602, ATCC 25619, and two clinical isolates (DFU-53 and 364707). In the presence of DHP, P. aeruginosa ATCC 25619 showed the highest decrease in pyocyanin production, whereas clinical isolate DFU-53 showed the lowest decrease. All three halogenated DHPs also reduced biofilm formation by between 31 and 34%. The non-halogenated compound 4d exhibited complete inhibition of LasR and had some inhibition of PqsR, pyocyanin, and biofilm formation, but comparatively less than halogenated DHPs.

Pyocyanin was the first natural phenazine described. The molecule is synthesized by about 95% of the strains of Pseudomonas aeruginosa. From discovery up to now, pyocyanin has been characterised by a very rich and avant-garde history, which includes its use in antimicrobial therapy, even before the discovery of penicillin opened the era of antibiotic therapy, as well as its use in electric current generation. Exhibiting an exuberant blue colour and being easy to obtain, this pigment is the subject of the present review, aiming to narrate its history as well as to unveil its mechanisms and suggest new horizons for applications in different areas of engineering, biology and biotechnology.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Electronic information can be transmitted to cells directly from microelectronics via electrode-activated redox mediators. These transmissions are decoded by redox-responsive promoters which enable user-specified control over biological function. Here, we build on this redox communication modality by establishing an electronic eCRISPR conduit of information exchange. This system acts as a biological signal processor, amplifying signal reception and filtering biological noise. We electronically amplify bacterial quorum sensing (QS) signaling by activating LasI, the autoinducer-1 synthase. Similarly, we filter out unintended noise by inhibiting the native SoxRS-mediated oxidative stress response regulon. We then construct an eCRISPR based redox conduit in both E. coli and Salmonella enterica. Finally, we display eCRISPR based information processing that allows transmission of spatiotemporal redox commands which are then decoded by gelatin-encapsulated E. coli. We anticipate that redox communication channels will enable biohybrid microelectronic devices that could transform our abilities to electronically interpret and control biological function.

Marine sponges, a well-documented prolific source of natural products, harbor highly diverse microbial communities. Their extracts were previously shown to contain quorum sensing (QS) signal molecules of the N -acyl homoserine lactone (AHL) type, known to orchestrate bacterial gene regulation. Some bacteria and eukaryotic organisms are known to produce molecules that can interfere with QS signaling, thus affecting microbial genetic regulation and function. In the present study, we established the production of both QS signal molecules as well as QS inhibitory (QSI) molecules in the sponge species Sarcotragus spinosulus . A total of eighteen saturated acyl chain AHLs were identified along with six unsaturated acyl chain AHLs. Bioassay-guided purification led to the isolation of two brominated metabolites with QSI activity. The structures of these compounds were elucidated by comparative spectral analysis of 1 HNMR and HR-MS data and were identified as 3-bromo-4-methoxyphenethylamine ( 1 ) and 5,6-dibromo- N , N -dimethyltryptamine ( 2 ). The QSI activity of compounds 1 and 2 was evaluated using reporter gene assays for long- and short-chain AHL signals ( Escherichia coli pSB1075 and E. coli pSB401, respectively). QSI activity was further confirmed by measuring dose-dependent inhibition of proteolytic activity and pyocyanin production in Pseudomonas aeruginosa PAO1. The obtained results show the coexistence of QS and QSI in S. spinosulus , a complex signal network that may mediate the orchestrated function of the microbiome within the sponge holobiont.

Biosurfactant - Rhamnolipids (RLs) and antibacterial toxin - pyocyanin (PYO) produced by Pseudomonas aeruginosa strains have great potential for biotechnological applications. Generally, RLs are produced as a mixture of di-rhamnolipids (di-RLs) and mono-rhamnolipids (mono-RLs). Mono-RLs possess superior emulsification and antimicrobial properties and are costlier than di-RLs. In this study, a taxonomic outlier P. aeruginosa strain CR1 isolated from rhizosphere soil was explored for mono-RLs and PYO production. Phylogenetically strain CR1 resembles avirulent outlier P. aeruginosa strain ATCC9027, lacks archetypical virulence genes and harbors unique pathways for the synthesis of solely mono-RLs and PYO. Strain CR1 produced RL biosurfactant which efficiently emulsified hydrocarbons, showed hemolysis and inhibited Bacillus subtilis. At 37 °C, strain CR1 exclusively produced 21.77 g L -1 and 19.22 g L -1 rhamnolipid in glycerol amended Luria Bertani (LB) medium and basal medium amended with rice bran oil, respectively after 54 h growth. Besides RL production was unaffected under varying nitrogen sources. Structural characterization using FTIR, TLC, and LC-MS confirmed that strain CR1 exclusively produced mono-RLs, majorly dominated by Rha-C 10 -C 10 , Rha-C 10 -C 8 , and CH 3 -Rha-C 12:2 -C 10:1 . The compound was stable over a wide pH range (4-12), salinity (25%) and 100 °C indicating its applicability under harsh environmental conditions. In addition, strain CR1 produced 4.5 μg mL -1 PYO, which could efficiently inhibit biofilm formation by Bacillus species. The environmental outlier strain CR1 can be used for the industrial production of biotechnologically important mono-RLs and PYO., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The stilbenoids, a group of naturally occurring phenolic compounds, are found in a variety of plants, including some berries that are used as food or for medicinal purposes. They are known to be beneficial for human health as anti-inflammatory, chemopreventive, and antioxidative agents. We have investigated a group of 19 stilbenoid substances in vitro using a cellular model of THP-1 macrophage-like cells and pyocyanin-induced oxidative stress to evaluate their antioxidant or pro-oxidant properties. Then we have determined any effects that they might have on the expression of the enzymes catalase, glutathione peroxidase, and heme oxygenase-1, and their effects on the activation of Nrf2. The experimental results showed that these stilbenoids could affect the formation of reactive oxygen species in a cellular model, producing either an antioxidative or pro-oxidative effect, depending on the structure pinostilbene ( 2 ) worked as a pro-oxidant and also decreased expression of catalase in the cell culture. Piceatannol ( 4 ) had shown reactive oxygen species (ROS) scavenging activity, whereas isorhapontigenin ( 18 ) had a mild direct antioxidant effect and activated Nrf2-antioxidant response element (ARE) system and elevated expression of Nrf2 and catalase. Their effects shown on cells in vitro warrant their further study in vivo., Competing Interests: The authors declare no conflict of interest.

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification., Competing Interests: No. The authors have declared that no competing interests exist.

In Pseudomonas aeruginosa ( Pae ), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d- arabino -heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. P. aeruginosa expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis. Here we report the structure of a type II DAH7PS, encoded by phzC as part of the duplicated phenazine biosynthetic cluster, from P. aeruginosa (PAO1) revealing for the first time the structure of a type II DAH7PS involved in secondary metabolism. The omission of the structural elements α 2a and α 2b , relative to other characterised type II DAH7PSs, leads to the formation of an alternative, dimeric, solution-state structure for this type II DAH7PS with an oligomeric interface that has not previously been characterised and that does not facilitate the formation of aromatic amino acid allosteric binding sites. The sequence similarity and, in particular, the common N-terminal extension suggest a common origin for the type II DAH7PSs from P. aeruginosa. The results described in the present study support an expanded classification of the type II DAH7PSs as type II A and type II B based on sequence characteristics, structure and function of the resultant proteins, and on defined physiological roles within primary or secondary metabolism., (© 2018 The Author(s).)

Bacterial quorum sensing systems regulate the production of an ample variety of bioactive extracellular compounds that are involved in interspecies microbial interactions and in the interplay between the microbes and their hosts. The development of new approaches for enabling chemical detection of such cellular activities is important in order to gain new insight into their function and biological significance. In recent years, surface-enhanced Raman scattering (SERS) spectroscopy has emerged as an ultrasensitive analytical tool employing rationally designed plasmonic nanostructured substrates. This review highlights recent advances of SERS spectroscopy for label-free detection and imaging of quorum sensing-regulated processes in the human opportunistic pathogen Pseudomonas aeruginosa . We also briefly describe the challenges and limitations of the technique and conclude with a summary of future prospects for the field.

The quorum sensing (QS) system has been used by many opportunistic pathogenic bacteria to coordinate their virulence determinants in relation to cell-population density. As antibiotic-resistant bacteria are on the rise, interference with QS has been regarded as a novel way to control bacterial infections. As such, many plant-based natural products have been widely explored for their therapeutic roles. These natural products may contain anti-QS compounds that could block QS signals generation or transmission to combat QS pathogens. In this study, we report the anti-QS activities of four different Chinese herbal plant extracts: Poria cum Radix pini , Angelica dahurica , Rhizoma cibotii and Schizonepeta tenuifolia , on Pseudomonas aeruginosa PAO1. All the plants extracted using hexane, chloroform and methanol were tested and found to impair swarming motility and pyocyanin production in P. aeruginosa PAO1, particularly by Poria cum Radix pini . In addition, all the plant extracts also inhibited violacein production in C. violaceum CV026 up to 50% while bioluminescence activities were reduced in lux -based E. coli biosensors, pSB401 and pSB1075, up to about 57%. These anti-QS properties of the four medicinal plants are the first documentation that demonstrates a potential approach to attenuate pathogens’ virulence determinants.

A bioelectrochemical sensing system based on a novel whole-cell redox reactivation/cycling module for ultrasensitive detection of pyocyanin (the biomarker of Pseudomonas aeruginosa infections) was developed. The electroactive bacteria mediated redox reactivation module was constructed using Shewanella oneidensis MR-1 cells as the bioelectro-catalyst and lactate as the electron donor. It could regenerate reductive pyocyanin from its oxidative state, which enabled pyocyanin molecule repeatedly registered by the electrode. Uniquely, with this redox reactivation module, the electrochemical response of pyocyanin was amplified about 405 times (1.3 μA/nM vs. 3.2nA/nM). Thus, an ultrasensitive bioelectrochemical sensing system for pyocyanin quantification was developed by integrating the pyocyanin reactivation module with conventional electrochemical detection system. Remarkably, with this developed biosensing system, an extremely low LOD of 47±1pM was reached. Additionally, this biosensing system showed excellent resistance to interferences from human fluids or bacterial contamination. This work provided a simple, ultrasensitive and robust tool for pyocyanin detection, and more importantly, demonstrated a new dimension for electrochemical signal amplification in biosensing., (Copyright © 2017 Elsevier B.V. All rights reserved.)

An ethyl acetate (EtOAc) extract isolated from the marine bacterium, Rheinheimera aquimaris QSI02, was found to exhibit anti-quorum sensing (anti-QS) activity. A subsequent bioassay-guided isolation protocol led to the detection of an active diketopiperazine factor, cyclo(Trp-Ser). Biosensor assay data showed that the minimum inhibitory concentration (MIC) of cyclo(Trp-Ser) ranged from 3.2 mg/ml to 6.4 mg/m for several microorganisms, including Escherichia coli, Chromobacterium violaceum CV026, Pseudomonas aeruginosa PA01, Staphylococcus aureus, and Candida albicans. Additionally, sub-MICs of cyclo(Trp-Ser) decreased the QS-regulated violacein production in C. violaceum CV026 by 67%. Furthermore, cyclo(Trp-Ser) can decrease QS-regulated pyocyanin production, elastase activity and biofilm formation in P. aeruginosa PA01 by 65%, 40% and 59.9%, respectively. Molecular docking results revealed that cyclo(Trp-Ser) binds to CviR receptor more rigidly than C 6 HSL with lower docking energy -8.68 kcal/mol, while with higher binding energy of -8.40 kcal/mol than 3-oxo-C 12 HSL in LasR receptor. Molecular dynamics simulation suggested that cyclo(Trp-Ser) is more easy to bind to CviR receptor than natural signaling molecule, but opposite in LasR receptor. These results suggest that cyclo(Trp-Ser) can be used as a potential inhibitor to control QS systems of C. violaceum and P. aeruginosa and provide increased the understanding of molecular mechanism that influences QS-regulated behaviors.

Pseudomonas aeruginosa can establish life-long chronic infection in patients with cystic fibrosis by generating genetic loss-of-function mutations, which enhance fitness of the bacterium in the airways. However, the precise role of the pathoadaptive mutations in persistence in chronic airways infection remains largely unknown. Here we demonstrate that pyocyanin, a well-described P. aeruginosa virulence factor that plays an important role in the initial infection, promotes autophagy in bronchial epithelial cells. Disruption of phzM, which is required for pyocyanin biosynthesis, leads to a significant reduction in autophagy in Beas-2B cells and lung tissues. Pyocyanin-induced autophagy is mediated by the EIF2AK4/GCN2-EIF2S1/eIF2α-ATF4 pathway. Interestingly, rats infected with the phzMΔ mutant strain have high mortality rate and numbers of colony-forming units, compared to those infected with wild-type (WT) P. aeruginosa PA14 strain, during chronic P. aeruginosa infection. In addition, the phzMΔ mutant strain induces more extensive alveolar wall thickening than the WT strain in the pulmonary airways of rats. As autophagy plays an essential role in suppressing bacterial burden, our findings provide a detailed understanding of why reduction of pyocyanin production in P. aeruginosa in chronic airways infections has been associated with better host adaptation and worse outcomes in cystic fibrosis.

Monitoring spatial distribution of metabolites in multicellular structures can enhance understanding of the biochemical processes and regulation involved in cellular community development. Here we report on an electrochemical camera chip capable of simultaneous spatial imaging of multiple redox-active phenazine metabolites produced by Pseudomonas aeruginosa PA14 colony biofilms. The chip features an 8 mm × 8 mm array of 1,824 electrodes multiplexed to 38 parallel output channels. Using this chip, we demonstrate potential-sweep-based electrochemical imaging of whole-biofilms at measurement rates in excess of 0.2 s per electrode. Analysis of mutants with various capacities for phenazine production reveals distribution of phenazine-1-carboxylic acid (PCA) throughout the colony, with 5-methylphenazine-1-carboxylic acid (5-MCA) and pyocyanin (PYO) localized to the colony edge. Anaerobic growth on nitrate confirms the O2-dependence of PYO production and indicates an effect of O2 availability on 5-MCA synthesis. This integrated-circuit-based technique promises wide applicability in detecting redox-active species from diverse biological samples.

Pseudomonas aeruginosa is an important human pathogen associated with several acute and chronic conditions, including diseases of the airways and wounds. The organism produces pyocyanin, an extracellular redox toxin that induces oxidative stress, depletes intracellular glutathione (GSH) and induces proliferative arrest and apoptosis, thus compromising the ability of tissue to repair itself. GSH is an important intra- and extracellular antioxidant, redox buffer and detoxifies xenobiotics by increasing their polarity, which facilitates their elimination. As previous studies have reported exogenous GSH to be protective against pyocyanin toxicity, this study was undertaken to explore the mechanism by which GSH protects host cells from the deleterious effects of the toxin. Co-incubation of pyocyanin with GSH resulted in a time-dependent diminished recovery of the toxin from the incubation medium. Concurrently, a highly polar green-colored metabolite was recovered that exhibited a UV-visible spectrum similar to pyocyanin and which was determined by mass spectrometry to have a major ion (m/z = 516) consistent with a glutathione conjugate. The ability of the conjugate to oxidize NADPH and to reduce molecular oxygen with the production of reactive oxygen species was comparable to pyocyanin yet it no longer demonstrated cytotoxicity towards host cells. These data suggest that GSH forms a cell-impermeant conjugate with pyocyanin and that availability of the thiol may be critical to minimizing the toxicity of this important bacterial virulence factor at infection sites. Our data indicate that for GSH to have a clinically effective role in neutralizing pyocyanin, the thiol needs to be available at millimolar concentrations., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation.

The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.

Bacteria are social organisms that possess multiple pathways for sensing and responding to small molecules produced by other microbes. Most bacteria in nature exist in sessile communities called biofilms, and the ability of biofilm bacteria to sense and respond to small molecule signals and cues produced by neighboring biofilm bacteria is particularly important. To understand microbial interactions between biofilms, it is necessary to perform rapid, real-time spatial quantification of small molecules in microenvironments immediately surrounding biofilms; however, such measurements have been elusive. In this study, scanning electrochemical microscopy was used to quantify small molecules surrounding a biofilm in 3D space. Measuring concentrations of the redox-active signaling molecule pyocyanin (PYO) produced by biofilms of the bacterium Pseudomonas aeruginosa revealed a high concentration of PYO that is actively maintained in the reduced state proximal to the biofilm. This gradient results in a reduced layer of PYO that we have termed the PYO "electrocline," a gradient of redox potential, which extends several hundred microns from the biofilm surface. We also demonstrate that the PYO electrocline is formed under electron acceptor-limiting conditions, and that growth conditions favoring formation of the PYO electrocline correlate to an increase in soluble iron. Additionally, we have taken a "reactive image" of a biofilm surface, demonstrating the rate of bacterial redox activity across a 2D surface. These studies establish methodology for spatially coordinated concentration and redox status measurements of microbe-produced small molecules and provide exciting insights into the roles these molecules play in microbial competition and nutrient acquisition.

The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.

The roles of the Pseudomonas aeruginosa-derived pigment pyocyanin (PYO) as an oxidant and activator of the proinflammatory transcription factor NF-kappaB were tested in a cystic fibrosis (CF) airway epithelial cell line, CF15. 100 microm PYO on its own had no effect or only small effects to activate NF-kappaB (<1.5-fold), but PYO synergized with the TLR5 agonist flagellin. Flagellin activated NF-kappaB 4-20-fold, and PYO increased these activations >2.5-fold. PYO could have synergized with flagellin to activate NF-kappaB by redox cycling with NADPH, generating superoxide (O(2)*), hydrogen peroxide (H(2)O(2)), and hydroxyl radical (HO*). Cytosol-targeted, redox-sensitive roGFP1 and imaging microscopy showed that 1-100 microm PYO oxidized CF15 cytosol redox potential (Psi(cyto)) from -325 mV (control) to -285 mV. O(2)* (derived from KO(2)*. or xanthine + xanthine oxidase) or H(2)O(2) oxidized Psi(cyto) dose-dependently but did not activate NF-kappaB, even in the presence of flagellin, and 400 microm H(2)O(2) inhibited NF-kappaB. Overexpressing intracellular catalase decreased effects of PYO and H(2)O(2) on Psi(cyto) but did not affect flagellin + PYO-activated NF-kappaB. Catalase also reversed the inhibitory effects of H(2)O(2) on NF-kappaB. The HO* scavenger DMSO did not alter the effects of PYO on Psi(cyto) and NF-kappaB. The synergistic NF-kappaB activation was calcium-independent. Thus, in the presence of flagellin, PYO activated NF-kappaB through a redox- and calcium-independent effect.

Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37 degrees C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O(2)-dependent. The formation of H(2)O(2) as an intermediate and the thiol group in GSH seem to be important in this reaction.

The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 A, alpha = gamma = 90, beta = 110.5 degrees. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 A. Seleno-L-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 A, alpha = gamma = 90, beta = 110.5 degrees and the diffraction limit is 2.7 A.

Pyocyanin, phenazine-1-carboxylic acid and more than 70 related compounds collectively known as phenazines are produced by various species of Pseudomonas, including the fluorescent pseudomonad P. aeruginosa, a Gram-negative opportunistic pathogen in humans and animals. P. aeruginosa synthesizes a characteristic blue water-soluble compound called pyocyanin (1-hydroxy-5-methyl-phenazine). Two enzymes designated PhzM and PhzS are involved in the terminal steps of its synthesis and very little is known about these enzymes. In this study, PhzM, a dimeric S-adenosylmethionine-dependent methyltransferase, was purified and crystallized from PEG 3350/sodium cacodylate/sodium citrate pH 6.5. The crystals belong to space group P1, with unit-cell parameters a = 46.1, b = 61.8, c = 69.6 A, alpha = 96.3, beta = 106.6, gamma = 106.9 degrees . They contain one dimer in the asymmetric unit and diffract to a resolution of 1.8 A. Anomalous data to 2.3 A resolution have been collected from seleno-L-methionine-labelled PhzM.

In several recent publications on pyocyanine, its mechanism of action has been attributed to an ability to react with nitric oxide (NO), resulting in the formation of an adduct. We examined the chemical interaction of pyocyanine and NO using electrospray (ES) MS, spectrophotometry and voltammetry at neutral pH and with 10-100 microM pyocyanine. No binding of NO to pyocyanine was observed. Alternative mechanisms for the inhibition of NO-induced vasorelaxation by pyocyanine should be sought.