Pyocyanin Facilitates Extracellular DNA Binding to Pseudomonas aeruginosa Influencing Cell Surface Properties and Aggregation

Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions.     

Pyocycanin,a Contributory Factor in Haem Acquisition and Virulence Enhancement of Porphyromonas gingivalis in the Lung

Several recent studies show that the lungs infected with Pseudomonas aeruginosa are often co-colonised by oral bacteria including black-pigmenting anaerobic (BPA) Porphyromonas species. The BPAs have an absolute haem requirement and their presence in the infected lung indicates that sufficient haem, a virulence up-regulator in BPAs, must be present to support growth. Haemoglobin from micro-bleeds occurring during infection is the most likely source of haem in the lung. Porphyromonas gingivalis displays a novel haem acquisition paradigm whereby haemoglobin must be firstly oxidised to methaemoglobin, facilitating haem release, either by gingipain proteolysis or capture via the haem-binding haemophore HmuY. P. aeruginosa produces the blue phenazine redox compound, pyocyanin. Since phenazines can oxidise haemoglobin, it follows that pyocyanin may also facilitate haem acquisition by promoting methaemoglobin production. Here we show that pyocyanin at concentrations found in the CF lung during P. aeruginosa infections rapidly oxidises oxyhaemoglobin in a dose-dependent manner. We demonstrate that methaemoglobin formed by pyocyanin is also susceptible to proteolysis by P. gingivalis Kgp gingipain and neutrophil elastase, thus releasing haem. Importantly, co-incubation of oxyhaemoglobin with pyocyanin facilitates haem pickup from the resulting methemoglobin by the P. gingivalis HmuY haemophore. Mice intra-tracheally challenged with viable P. gingivalis cells plus pyocyanin displayed increased mortality compared to those administered P. gingivalis alone. Pyocyanin significantly elevated both methaemoglobin and total haem levels in homogenates of mouse lungs and increased the level of arginine-specific gingipain activity from mice inoculated with viable P. gingivalis cells plus pyocyanin compared with mice inoculated with P. gingivalis only. These findings indicate that pyocyanin, by promoting haem availability through methaemoglobin formation and stimulating of gingipain production, may contribute to virulence of P. gingivalis and disease severity when co-infecting with P. aeruginosa in the lung.     

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis‐like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin‐elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca²⁺ activity as well as Ca²⁺‐dependent proteolytic processing of μ‐calpain. Pyocyanin further up‐regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin‐induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl‐ester labelling, pyocyanin‐treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis‐inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.     

A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity.

Pyocyanin was prepared in 60% yield from phenazine methoxysulfate by a photooxidation procedure and purification by silica gel chromatography. Monitoring was performed by thin-layer chromatography. Approximately 50% of clinical Pseudomonas aeruginosa isolates were found to produce pyocyanin at 37°C. Among Proteus strains, P. morganii strains were sensitive to concentrations of pyocyanin 16 to 64 times lower than concentrations that inhibited the growth of P. mirabilis and P. vulgaris strains.     

Metabolic engineering of E. coli for pyocyanin production

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.     

Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans

Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.     

Effect of Pyocyanin on a Crude-Oil-Degrading Microbial Community

Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 μM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C₂₉ and C₃₀ hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.     

Pyocyanin induced in vitro oxidative damage and its toxicity level in human,fish and insect cell lines for its selective biological applications

Pyocyanin is a redox active phenazine pigment produced by Pseudomonas aeruginosa, with broad antibiotic activity having pharmacological, aquaculture, agriculture and industrial applications. In the present work cytotoxicity induced by pyocyanin is demonstrated in a human embryonic lung epithelial cell line (L-132), a rainbow trout gonad cell line (RTG-2) and a Spodoptera frugiperda pupal ovarian cell line (Sf9). For toxicity evaluation, cellular morphology, mitochondrial function (XTT), membrane leakage of lactate dehydrogenase, neutral red uptake, affinity of electrostatic binding of protein with sulforhodamine B dyes, glucose metabolism, and reactive oxygen species, were assessed. Results showed that higher pyocyanin concentration is required for eliciting cytotoxicity in L-132, RTG-2 and Sf9. The microscopic studies demonstrated that the cell lines exposed to pyocyanin at higher concentrations alone showed morphological changes such as clumping and necrosis. Among the three cell lines L-132 showed the highest response to pyocyanin than the others. In short, pyocyanin application at concentrations ranging from 5 to 10 mg l^?1 were not having any pathological effect in eukaryotic systems and can be used as drug of choice in aquaculture against vibrios in lieu of conventional antibiotics and as biocontrol agent against fungal and bacterial pathogens in agriculture. This is besides its industrial and pharmacological applications.     

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

Pseudomonas aeruginosa-Candida albicans Interactions: Localization and Fungal Toxicity of a Phenazine Derivative

Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.     

Sodium houttuyfonate, a potential phytoanticipin derivative of antibacterial agent, inhibits bacterial attachment and pyocyanine secretion of Pseudomonas aeruginosa by attenuating flagella-mediated swimming motility

Pseudomonas aeruginosa is a well-known clinical pathogen for its recalcitrant infection caused by biofilm formation which are initiated by flagella-mediated attachment. Sodium houttuyfonate (SH) is a natural phytoanticipin derivative of houttuynin and has anti-pathogenic effect on P. aeruginosa biofilm formation. In this paper, when using 1/2 × MIC SH, the diameter of P. aeruginosa swimming motility was sharply shortened to 36 % in 24 h incubation, and the fold changes of fliC required for swimming motility was 0.36 in 24 h cultivation, the adherence inhibition accounted for about 46 %, and the pyocyanin production decreased to 47 % after 1-day treatment and 56 % after 3-day treatment with obvious visual changes from dark green to light green, compared with the negative control. With the help of mass spectra and scanning electronic microscope, 1/2 × MIC SH was further testified to be enough to eradicate flagella and inhibit pyocyanin secretion of P. aeruginosa. The results do not only re-affirm the close interplay of attachment and virulence (i.e. swimming motility and pyocyanin), but also unravel the potential mechanism of SH on anti-biofilm of P. aeruginosa.     

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation

Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.     

Oxidation of pyocyanin, a cytotoxic product from Pseudomonas aeruginosa, by microperoxidase 11 and hydrogen peroxide

Pyocyanin (1-hydroxy-N-methylphenazine) is a cytotoxic pigment secreted by the bacterial species Pseudomonas aeruginosa, which frequently infects the lungs of immunosuppressed patients as well as those with cystic fibrosis. Pyocyanin toxicity results presumably from the ability of the compound to undergo reduction by NAD(P)H and subsequent generation of superoxide and H2O2 directly in the lungs. We report that in the presence of peroxidase mimics, microperoxidase 11, or hemin, pyocyanin undergoes oxidation by H2O2, as evidenced by loss of the pigment's characteristic absorption spectrum and by EPR detection of a free radical metabolite. The oxidation of pyocyanin is irreversible, suggesting an extensive modification of the pigment's phenazine chromophore. Oxidation of pyocyanin was observed also when exogenous H2O2 was replaced by a H2O2-generating system consisting of NADH and the pigment itself. That the oxidation involves the phenolate group of pyocyanin was verified by the observation that a related pigment, phenazine methosulfate, which is devoid of this group, does not undergo oxidation by microperoxidase 11/H2O2. In contrast to intact pyocyanin, oxidized pyocyanin was less efficient in NADH oxidation and stimulation of interleukin-8 release by human alveolar epithelial A549 cells in vitro, suggesting that oxidation of pyocyanin leads to its inactivation. This study demonstrates that pyocyanin may play a dual role in biological systems, first as an oxidant and ROS generator, and second as a substrate for peroxidases, contributing to H2O2 removal. This latter property may cause pyocyanin degradation and inactivation, which may be of considerable biomedical interest.     

Optimization of nutritional and environmental conditions for pyocyanin production by urine isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacteria involved in numerous diseases among which, are urinary tract infections (UTIs). The pyocyanin secreted as a virulence factor by this bacterium has many beneficial applications but its high cost remains an obstacle for its widespread use. In this study, a total of fifty urine isolates were identified as P. aeruginosa. All strains produced pyocyanin pigment with a range of 1.3–31 µg/ml. The highest producer clinical strain P21 and the standard strain PA14 were used in optimization of pyocyanin production. Among tested media, king’s A fluid medium resulted in the highest yield of pyocyanin pigment followed by nutrient broth. Growth at 37 °C was superior in pyocyanin production than growth at 30 °C. Both shaking and longer incubation periods (3–4 days) improved pyocyanin production. The pyocyanin yield was indifferent upon growth of P21 at both pH 7 and pH 8. In conclusion, the optimum conditions for pyocyanin production are to use King’s A fluid medium of pH 7 and incubate the inoculated medium at 37 °C with shaking at 200 rpm for a period of three to four days.     

Inhibition of biofilm formation,quorum sensing activity and molecular docking study of isolated 3, 5, 7-Trihydroxyflavone from Alstonia scholaris leaf against P.aeruginosa

The current study is to evaluate the inhibition of biofilm formation and quorum sensing activity of isolated 3, 5, 7-Trihydroxyflavone (TF) from A.scholaris leaf extract against Pseudomonas aeruginosa. The effects of isolated TF on quorum sensing-regulated virulence factors production such as swimming motility, pyocyanin production, proteolytic, EPS, metabolic assay and inhibition of biofilm formation against P.aeruginosa was evaluated by standard protocols. In addition, the interaction between the isolated TF and active sites of QS- gene (LasI/rhlI, LasR/rhlR, and AHLase) in P.aeruginosa was evaluated by molecular docking studies using AutoDock Tools version 1.5.6. Based on the structural elucidation of the isolated compound was identified as 3, 5, 7-Trihydroxyflavone. Consequently, the isolated TF shows a significant reduction of biofilm formation through the inhibition of QS-dependent phenotypes such as pyocyanin production, proteolytic, swimming motility, EPS activities against P.aeruginosa in a dose-dependent manner. Molecular docking analysis of isolated TF can interfere the signaling [N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL)] molecules in P.aeruginosa by QS genes (LasI, LasR, rhlI, and AHLase) regulation. The isolated TF compound from A.scholaris reveals a greater potential to inhibit biofilm and QS dependent virulence factor production in P.aeruginosa. Docking interaction studies of TF-LasR complex express higher binding affinity than the other QS gene in P.aeruginosa.     

Glutathione modulates the toxicity of, but is not a biologically relevant reductant for, the Pseudomonas aeruginosa redox toxin pyocyanin

Pyocyanin is an important redox toxin produced by the common human pathogen Pseudomonas aeruginosa. It generates reactive oxygen species (ROS) that alter intracellular redox status and cell function. Reducing equivalents for pyocyanin are provided by intracellular NAD(P)H and, it has been reported, glutathione (GSH). Cellular GSH levels are at least 1-2 orders of magnitude greater than NAD(P)H; therefore GSH should represent the major reductant for pyocyanin and potentiate its toxicity. Paradoxically, GSH has been found to inhibit pyocyanin toxicity in cellular models. This study was undertaken to evaluate the potential of GSH as a biologically relevant reductant for pyocyanin. As observed using spectrophotometry, under aerobic conditions pyocyanin readily oxidized NADPH, whereas oxidation of GSH could not be detected. Under anaerobic conditions pyocyanin was reduced by NADPH, but reduction by GSH could not be detected. Reduction of molecular oxygen and the formation of ROS readily proceeded in the presence of pyocyanin and NADPH, whereas GSH was without effect. Finally, exposure of normal human dermal fibroblasts to subcytotoxic concentrations of pyocyanin did not lead to depletion of endogenous GSH, but exogenous GSH provided protection against the senescence-inducing effects of the toxin. In summary, GSH does not reduce pyocyanin under physiologically relevant conditions or contribute to pyocyanin toxicity. However, GSH does provide protection against the deleterious effects of this important bacterial toxin on mammalian cells.     

Premature cellular senescence induced by pyocyanin, a redox-active Pseudomonas aeruginosa toxin

Pseudomonas aeruginosa is an important nosocomial pathogen that can cause acute and chronic infection, particularly of the respiratory system. Pyocyanin is a major P. aeruginosa virulence factor that displays redox activity and induces oxidative stress in cellular systems. The effect of pyocyanin on replicating human pulmonary epithelial (A549) cells was investigated. Cells were exposed to pyocyanin for 24 h and their subsequent growth and development were followed for 7 days. Pyocyanin (5-10 microM) arrested cell growth and resulted in the development of a morphological phenotype consistent with cellular senescence, that is, an enlarged and flattened appearance. The senescent nature of these cells was supported by positive staining for increased lysosomal content and senescence-associated beta-galactosidase activity. All cells treated with pyocyanin (10 microM) converted to the senescent phenotype, which remained stable for up to 7 days. Exposure to pyocyanin at 25 microM or greater resulted in cell death due to apoptosis. A549 cells exposed to pyocyanin generated hydrogen peroxide in a dose-dependent manner and the senescence-inducing effect of pyocyanin was inhibited by the antioxidant, glutathione, suggesting the involvement of reactive oxygen species. The induction of premature cellular senescence by redox-active bacterial toxins may be a hitherto unrecognized aspect of infection pathology and a limiting factor in the tissue repair response to infection.     

Loss of Social Behaviours in Populations of Pseudomonas aeruginosa Infecting Lungs of Patients with Cystic Fibrosis

Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections.     

A common evolutionary pathway for maintaining quorum sensing in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In the bacterium Pseudomonas aeruginosa, the synthesis and secretion of extracellular protease is a typical cooperative behavior regulated by quorum sensing. However, this type of cooperative behavior is easily exploited by other individuals who do not synthesize public goods, which is known as the “tragedy of the commons”. Here P. aeruginosa was inoculated into casein media with different nitrogen salts added. In casein broth, protease (a type of public good) is necessary for bacterial growth. After 30 days of sequential transfer, some groups propagated stably and avoided “tragedy of the commons”. The evolved cooperators who continued to synthesize protease were isolated from these stable groups. By comparing the characteristics of quorum sensing in these cooperators, an identical evolutionary pattern was found. A variety of cooperative behaviors regulated by quorum sensing, such as the synthesis and secretion of protease and signals, were significantly reduced during the process of evolution. Such reductions improved the efficiency of cooperation, helping to prevent cheating. In addition, the production of pyocyanin, which is regulated by the RhlIR system, increased during the process of evolution, possibly due to its role in stabilizing the cooperation. This study contributes towards our understanding of the evolution of quorum sensing of P. aeruginosa.