Universal antibiotic tolerance arising from antibiotic-triggered accumulation of pyocyanin in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects open wounds or patients with cystic fibrosis. Once established, P. aeruginosa infections are notoriously difficult to eradicate. This difficulty is in part due to the ability of P. aeruginosa to tolerate antibiotic treatment at the individual-cell level or through collective behaviors. Here, we describe a new phenomenon by which P. aeruginosa tolerates antibiotic treatment. In particular, treatment of P. aeruginosa with sublethal concentrations of antibiotics covering all major classes promoted accumulation of the redox-sensitive phenazine pyocyanin (PYO). PYO in turn conferred general tolerance against diverse antibiotics for both P. aeruginosa and other gram-negative and gram-positive bacteria. This property is shared by other redox-active phenazines produced by P. aeruginosa. Our discovery sheds new insights into the physiological functions of phenazines and has implications for designing effective antibiotic treatment protocols.

The bacterial pigment pyocyanin inhibits the NLRP3 inflammasome through intracellular reactive oxygen and nitrogen species.

Inflammasomes are cytosolic complexes that mature and secrete the inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18 and induce pyroptosis. The NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome detects many pathogen- and danger-associated molecular patterns, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) have been implicated in its activation. The phenazine pyocyanin (PCN) is a virulence factor of Pseudomonas aeruginosa and generates superoxide in cells. Here we report that PCN inhibits IL-1ß and IL-18 release and pyroptosis upon NLRP3 inflammasome activation in macrophages by preventing speck formation and Caspase-1 maturation. Of note, PCN did not regulate the AIM2 (absent in melanoma 2) or NLRC4 inflammasomes or tumor necrosis factor (TNF) secretion. Imaging of the fluorescent glutathione redox potential sensor Grx1-roGFP2 indicated that PCN provokes cytosolic and nuclear but not mitochondrial redox changes. PCN-induced intracellular ROS/RNS inhibited the NLRP3 inflammasome posttranslationally, and hydrogen peroxide or peroxynitrite alone were sufficient to block its activation. We propose that cytosolic ROS/RNS inhibit the NLRP3 inflammasome and that PCN's anti-inflammatory activity may help P. aeruginosa evade immune recognition.

Cellular Effects of Pyocyanin, a Secreted Virulence Factor of Pseudomonas aeruginosa.

Pyocyanin has recently emerged as an important virulence factor produced by Pseudomonas aeruginosa. The redox-active tricyclic zwitterion has been shown to have a number of potential effects on various organ systems in vitro, including the respiratory, cardiovascular, urological, and central nervous systems. It has been shown that a large number of the effects to these systems are via the formation of reactive oxygen species. The limitations of studies are, to date, focused on the localized effect of the release of pyocyanin (PCN). It has been postulated that, given its chemical properties, PCN is able to readily cross biological membranes, however studies have yet to be undertaken to evaluate this effect. This review highlights the possible manifestations of PCN exposure; however, most studies to date are in vitro. Further high quality in vivo studies are needed to fully assess the physiological manifestations of PCN exposure on the various body systems.

Immunochemical Determination of Pyocyanin and 1-Hydroxyphenazine as Potential Biomarkers of Pseudomonas aeruginosa Infections.

A novel immunochemical approach to diagnose Pseudomonas aeruginosa infections is reported, which is based on the quantification of relevant and specific virulence factors secreted by this microorganism. Specific antibodies have been raised using hapten PC1 (a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids), designed to recognize 1-hydroxyphenazine (1-OHphz), which is the main metabolite of pyocyanin (PYO). PYO is one of the most important virulence factors produced by nearly all P. aeruginosa strains, and other species do not produce this factor. With these antibodies, an immunochemical analytical procedure able to quantify both 1-OHphz and PYO in complex clinical samples has been developed. 1-OHphz can be directly measured in solubilized sputum samples diluted 20 times with the assay buffer. Quantification of PYO is accomplished after conversion to 1-OHphz in just 20 min under basic conditions. A LOD of 0.60 ± 0.01 nM (4.80 ± 0.08 nmol kg(-1) sputum) is reached for both biomarker targets under the conditions established, a value that is much below the reported concentrations on sputum samples obtained from infected patients (up to 100 µM). The assay is robust, reproducible, accurate, can be run in about 2 h, and many samples can be measured simultaneously. The present reported assay could represent a significant improvement in the diagnosis of infectious diseases caused by this pathogen.

Pseudomonas pyocyanin stimulates IL-8 expression through MAPK and NF-κB pathways in differentiated U937 cells.

BACKGROUND: Pyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox active secondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatory response triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to be clarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction and the signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined. RESULTS: It was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA) differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after 10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also found that within 10 min of PCN incubation, the level of p-I-κBα in the cytosol was increased, which returned to baseline level after 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high in total cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-κB inhibitors inhibited PCN-induced NF-κB activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of their concentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine, an antioxidant, was able to inhibit PCN-induced IL-8 protein expression. CONCLUSIONS: It is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937 cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF-κΒ signaling pathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.

Differential potentiation of the virulence of the Pseudomonas aeruginosa cystic fibrosis liverpool epidemic strain by oral commensal Streptococci.

BACKGROUND: The Pseudomonas aeruginosa Liverpool epidemic strain (LES) is an important cystic fibrosis (CF) pathogen and is associated with increased morbidity and a worsened prognosis, compared with other CF-associated strains. However, interactions of common LES phenotypic variants with other members of the polymicrobial biofilms associated with chronic CF respiratory disease, such as oral commensal streptococci, have not been investigated. METHODS: Biofilm population dynamics, virulence factor production, and pathogenicity in Galleria mellonella larvae of common LES phenotypes (ie, low production, intermediate production, and overproduction of pyocyanin) in the presence or absence of anginosus group streptococci (AGS) were compared. RESULTS: AGS populations isolated from biofilm cocultures were P. aeruginosa phenotypic variant dependent, with higher AGS cell densities than those in monoculture frequently observed. Coexistence of AGS with a producer of low or intermediate levels of pyocyanin was found to result in enhancement of virulence factor production. In addition, the LES formed pathogenic partnerships with AGS in the G. mellonella infection model, with killing dependent on LES phenotype and AGS species. CONCLUSIONS: The pathogenic potential of LES phenotypic variants can be enhanced by the presence of oral commensal streptococci. As adaptive mutations leading to reduced virulence factor production are commonplace, the observations made are relevant in the general context of the biology of P. aeruginosa infection during CF.

Redox warfare between airway epithelial cells and Pseudomonas: dual oxidase versus pyocyanin.

The importance of reactive oxygen species-dependent microbial killing by the phagocytic cell NADPH oxidase has been appreciated for some time, although only recently has an appreciation developed for the partnership of lactoperoxidase with related dual oxidases (Duox) within secretions of the airway surface layer. This system produces mild oxidants designed for extracellular killing that are effective against several airway pathogens, including Staphylococcus aureus, Burkholderia cepacia, and Pseudomonas aeruginosa. Establishment of chronic pseudomonas infections involves adaptations to resist oxidant-dependent killing by expression of a redox-active virulence factor, pyocyanin, that competitively inhibits epithelial Duox activity by consuming intracellular NADPH and producing superoxide, thereby inflicting oxidative stress on the host.

Similar C-phycocyanins from two strains of thermotolerant cyanophyte Mastigocladus laminousus.

C-phycocyanin from two strains of the thermotolerant blue-green alga, Mastigocladus laminosus (NZ-DB2-m and I-30-m), that grow within different temperature ranges have been characterized with respect to aggregation, immunologic properties, subunit composition, and thermodenaturation. The critical thermal-denaturation temperature for phycocyanin from both strains of M. laminosus phycocyanin is 60 degrees C which is higher than that for mesophilic phyococyanin. Immunodiffusion studied have shown that these two strains of M. laminosus exhibit no antigenic differences and are closely related to the mesophilic Plectonema calothricoides and the thermophilic Synechococcus lividus (strains 3). Neither phenol nor alpha-naphthol has any effect on phycocyanin aggregation in these two strains of M. laminosus. There is also no enhancement of formation of large aggregates at their elevated temperature of cultivation. Furthermore, the phycocyanin of both strains of M. laminosus does not demonstrate any large amount of 19S or higher aggregates at any pH value. These observations suggest that the mode of adaptation of M. laminosus phycocyanin to high temperature is differnet from the previously encountered. It is also important to note that phycocyanin is essentially unchanged whether it is extracted from the same strain, M. laminosus (NZ-DBS-m), grown at either 50 degrees C or 37 degrees C.