Pyocyanin Treatment Research Articles

Antibacterial, antibiofilm, and anti-quorum sensing activities of pyocyanin against methicillin-resistant Staphylococcus aureus: in vitro and in vivo study

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) infections are considered a major public health problem, as the treatment options are restricted. Biofilm formation and the quorum sensing (QS) system play a pivotal role in S. aureus pathogenicity. Hence, this study was performed to explore the antibacterial effect of pyocyanin (PCN) on MRSA as well as its effect on MRSA biofilm and QS.ResultsData revealed that PCN exhibited strong antibacterial activity against all test MRSA isolates (n = 30) with a MIC value equal to 8 µg/ml. About 88% of MRSA biofilms were eradicated by PCN treatment using the crystal violet assay. The disruption of MRSA biofilm was confirmed using confocal laser scanning microscopy, which showed a reduction in bacterial viability (approximately equal to 82%) and biofilm thickness (approximately equal to 60%). Additionally, the disruption of the formation of microcolonies and the disturbance of the connection between bacterial cells in the MRSA biofilm after PCN treatment were examined by scanning electron microscopy. The 1/2 and 1/4 MICs of PCN exerted promising anti-QS activity without affecting bacterial viability; Agr QS-dependent virulence factors (hemolysin, protease, and motility), and the expression of agrA gene, decreased after PCN treatment. The in silico analysis confirmed the binding of PCN to the AgrA protein active site, which blocked its action. The in vivo study using the rat wound infection model confirmed the ability of PCN to modulate the biofilm and QS of MRSA isolates.ConclusionThe extracted PCN seems to be a good candidate for treating MRSA infection through biofilm eradication and Agr QS inhibition.

Glucocorticoid receptors involved in melatonin inhibiting cell apoptosis and NLRP3 inflammasome activation caused by bacterial toxin pyocyanin in colon

The immunoinhibitory effect of glucocorticoid and immunoenhancing attributes of melatonin (MEL) are well known, however, the involvement of glucocorticoid receptor (GR) in melatonin modulation of bacterial toxins caused-inflammation has not been studied in colon. Pyocyanin (PCN), a toxin released by Pseudomonas aeruginosa, can destroy cells through generating superoxide products and inflammatory response. Here we report that PCN treatment elevated the generation of reactive oxygen species (ROS), which further lead to mitochondrial swelling and caspase cascades activation both in vivo and in vitro. However, MEL treatment alleviated the oxidative stress caused by PCN on cells through scavenging ROS and restoring the expression of antioxidant enzyme so that to effectively alleviate the apoptosis. Large amounts of ROS can activate the NLRP3 signaling pathway, so MEL inhibited PCN induced NLRP3 inflammasome activation and inflammatory cytokines (IL-1β, IL-8, and TNF-α) secretion. In order to further investigate the molecular mechanism, goblet cells were exposed to MEL and PCN in the presence of luzindole and RU486, inhibitors of MEL receptors and GR respectively. It was found that PCN significantly inhibited the expression level of GR, and MEL effectively alleviated the inhibition phenomenon. Moreover, we found that MEL mainly upregulated the expression of GR to achieve its anti-inflammatory and anti-apoptotic functions rather than through its own receptor (MT2) in colon goblet cells. Therefore, MEL can reverse the inhibitory effects of PCN on GR/p-GR expression to present its anti-oxidative and anti-apoptotic function.

Antioxidation and Antiapoptosis Characteristics of Heme Oxygenase-1 Enhance Tumorigenesis of Human Prostate Carcinoma Cells

Heme oxygenase-1 (HO-1) has antiinflammatory and antioxidant properties and is deemed as a tissue protector. However, effects of HO-1 in prostate cancer remain in controversy. We evaluated the role of HO-1 in prostate carcinoma in vitro and in vivo.Overexpression of HO-1 did not affect prostate cell proliferation in the normal condition but enhanced cell proliferation under serum starvation. HO-1 overexpression enhanced cell invasion of PC-3 cells through epithelial–mesenchymal transition (EMT) induction, which was supported by increased Slug, N-cadherin, and vimentin expressions. In the xenograft animal study, HO-1 overexpression enhanced PC-3 cell tumor growth in vivo. HO-1 attenuated reactive oxygen species induced by H2O2 or pyocyanin treatment in PC-3 and DU145 cells. HO-1 further reduced PC-3 and DU145 cell apoptosis induced by H2O2 or serum starvation. Our results suggested that HO-1 was able to increase prostate carcinoma cell invasion in vitro and tumor growth in vivo. The EMT induction and antioxidant and antiapoptotic effects of HO-1 in the prostate carcinoma cells may be responsible for these findings.

Cytotoxic effect of pyocyanin on human pancreatic cancer cell line (Panc-1).

Objective(s):Pyocyanin, a blue-green pigment produced by Pseudomonas aeruginosa, interferes with host redox cycles, which can lead to generation of reactive oxygen species and progressive cellular oxidative damage. The aim of this study was to assess the influence of pyocyanin on human pancreatic cancer cell line.Materials and Methods:Polymerase Chain Reaction (PCR) was applied to confirm the existence of a specific pyocyanin producing gene (phzM). The pigment was then characterized by UV-visible, FT-IR, and HPLC analysis. Panc-1 cells were treated by different concentrations of pyocyanin and their cytotoxic effect as well as the induction of apoptosis/necrosis were evaluated by XTT assay and flow cytometry.Results:An overnight pyocyanin treatment resulted in significant cell reduction in a concentration-dependent manner. Inhibition rate of 6 mg.ml-1 pyocyanin (the highest concentration) extracted from clinical and soil isolates of P. aeruginosa were 98.69±0.23 and 89.88±1.86%, respectively, which decreased as the pyocyanin concentration lessened. Pyocyanin could also induce dose-dependent apoptosis/necrosis in Panc-1 cells after 24 hr.Conclusion:We reported, for the first time, cytotoxic effects of pyocyanin against human pancreatic cancer cell line. Considering this effect of the pigment, study on pyocyanin as a potential anti-tumor biodrug requires further studies.

Anticancer effects of pyocyanin on HepG2 human hepatoma cells

Pyocyanin, a major virulence factor produced by Pseudomonas aeruginosa, displays redox activity and damaging effects on mammalian cells. In this study, we investigated the effects of pyocyanin on the proliferation of HepG2 tumour cells. Interestingly, pyocyanin significantly inhibited cell proliferation and triggered the production of large amounts of reactive oxygen species (ROS), thereby upregulating superoxide dismutase (SOD) and catalase (CAT). Additionally, pyocyanin treatment significantly depleted reduced glutathione (GSH) and decreased the GSH/oxidized GSH (GSSG) ratio. These results supported that pyocyanin-induced cytotoxicity in HepG2 cells was mediated by acute ROS production and subsequent oxidative stress. SA-β-Gal, acridine orange (AO)/ethidium bromide (EB) double staining, caspase-3 measurements and comet assay results revealed that cell death induced by pyocyanin involved DNA damage and activation of caspase-3, accelerating cell senescence and apoptosis. Thus, our data provided insights into the mechanisms underlying pyocyanin-induced cytotoxicity and may lead to better treatment strategies for cancer. Pyocyanin is a redox-active phenazine toxin. Here, we investigated the ability of pyocyanin to inhibit cancer-related phenotypes in HepG2 human hepatoma cells. Our results indicated that pyocyanin accelerated cellular senescence and apoptosis and induced oxidative stress-associated DNA damage in HepG2 cells. The potential anticancer applications of pyocyanin should be investigated further in clinical studies.

Effects of Pseudomonas Aeruginosa Virulence Factor Pyocyanin on Human Urothelial Cell Function and Viability

We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection.

Liver sinusoidal endothelial cells and acute non‐oxidative hepatic injury induced by Pseudomonas aeruginosa pyocyanin

The liver sinusoidal endothelial cell (LSEC) is damaged by many toxins, including oxidants and bacterial toxins. Any effect on LSECs of the Pseudomonas aeruginosa virulence factor, pyocyanin, may be relevant for systemic pseudomonal infections and liver transplantation. In this study, the effects of pyocyanin on in vivo rat livers and isolated LSECs were assessed using electron microscopy, immunohistochemistry and biochemistry. In particular, the effect on fenestrations, a crucial morphological aspect of LSECs was assessed. Pyocyanin treatment induced a dose-dependent reduction in fenestrations in isolated LSECs. In the intact liver, intraportal injection of pyocyanin (11.9 microM in blood) was associated with a reduction in endothelial porosity from 3.4 +/- 0.2% (n = 5) to 1.3 +/- 0.1% (n = 7) within 30 min. There were decreases in both diameter and frequency of fenestrations in the intact endothelium. There was also a decrease in endothelial thickness from 175.8 +/- 5.8 to 156.5 +/- 4.0 nm, an endothelial pathology finding previously unreported. Hepatocyte ultrastructure, liver function tests and immunohistochemical markers of oxidative stress (3-nitrotyrosine and malondialdehyde) were not affected. Pyocyanin induces significant ultrastructural changes in the LSEC in the absence of immunohistochemical evidence of oxidative stress or hepatocyte injury pointing to a novel mechanism for pyocyanin pathogenesis.

Subversion of a Lysosomal Pathway Regulating Neutrophil Apoptosis by a Major Bacterial Toxin, Pyocyanin

Abstract Neutrophils undergo rapid constitutive apoptosis that is accelerated following bacterial ingestion as part of effective immunity, but is also accelerated by bacterial exotoxins as a mechanism of immune evasion. The paradigm of pathogen-driven neutrophil apoptosis is exemplified by the Pseudomonas aeruginosa toxic metabolite, pyocyanin. We previously showed pyocyanin dramatically accelerates neutrophil apoptosis both in vitro and in vivo, impairs host defenses, and favors bacterial persistence. In this study, we investigated the mechanisms of pyocyanin-induced neutrophil apoptosis. Pyocyanin induced early lysosomal dysfunction, shown by altered lysosomal pH, within 15 min of exposure. Lysosomal disruption was followed by mitochondrial membrane permeabilization, caspase activation, and destabilization of Mcl-1. Pharmacological inhibitors of a lysosomal protease, cathepsin D (CTSD), abrogated pyocyanin-induced apoptosis, and translocation of CTSD to the cytosol followed pyocyanin treatment and lysosomal disruption. A stable analog of cAMP (dibutyryl cAMP) impeded the translocation of CTSD and prevented the destabilization of Mcl-1 by pyocyanin. Thus, pyocyanin activated a coordinated series of events dependent upon lysosomal dysfunction and protease release, the first description of a bacterial toxin using a lysosomal cell death pathway. This may be a pathological pathway of cell death to which neutrophils are particularly susceptible, and could be therapeutically targeted to limit neutrophil death and preserve host responses.

Mechanisms of action of Pseudomonas aeruginosa pyocyanin on human ciliary beat in vitro.

Pyocyanin is a blue redox active pigment produced by Pseudomonas aeruginosa. It is present at concentrations of up to 10(-4) M in sputa from patients with cystic fibrosis and bronchiectasis who are heavily colonized with this organism. Pyocyanin, at physiologically relevant concentrations, slows human nasal ciliary beat frequency (CBF) in vitro and leads to disruption of the epithelium. Pyocyanin-induced slowing of CBF after 2 h was associated with a significant fall in intracellular cyclic AMP (cAMP) (90%) and ATP (66%) and was reversible after the pyocyanin was removed by washing. These effects were not mediated through interaction with neutrophils. The pyocyanin-induced fall in CBF was not affected by EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], pyrazinamide, 8-phenyltheophylline, indomethacin, or antioxidants, including catalase (500 U/ml), superoxide dismutase, and N-acetylcysteine. Ciliary slowing was, however, prevented (> 70%) by isobutylmethylxanthine and forskolin, both of which increase intracellular cAMP, and also by the cAMP analog, dibutyryl cAMP. There was also a concomitant protection against the fall in both cAMP and ATP. These agents also delayed the onset of epithelial disruption associated with pyocyanin treatment. In contrast, treatment with the iron chelator desferrioxamine prevented epithelial disruption, although it had no effect on pyocyanin-induced slowing of CBF. It appears that ciliary slowing can be dissociated from epithelial disruption and that the effects of pyocyanin on CBF are associated with a fall in both intracellular cAMP and ATP.