Pseudomonas aeruginosa produces copious amounts of the redox-active tricyclic phenazine compound pyocyanin ( chloroform-soluble) that kills competing microbes and mammalian cells, especially during cystic fibrosis ,lung infection. Pyocyanin is responsible for the blue-green colour chacteristics of pseudomonas species. The pigment pyocyanin is composed of 2 subunits of N-methyl-1-hydroxyphenazine . Pyocyanin has a molecular weight of approximately 210.27 grams per mole and its chemical structure consists of a pyrrole ring with a phenazine group attached as shown in it’s structure. Pyocyanin has been crystallized in pure form and classified as a phenazine-type of molecule . The phenazine-based pyocyanin pigment has a particular interest for its capability to generate reactive oxygen species (ROS). Tumor cells are susceptible to reactive oxygen species produced by pyocyanin since it interferes with topoisomerase I and II activities in eukaryotic cells.
The mechanism of action is that having a low molecular weight of 210 Da zwitterion, pyocyanin can be easily permeable through the cell membrane, and being a redox-active biomolecule, it can regulate the generation of oxidative stress by increasing the intracellular levels of reactive oxygen species (ROS) and hydrogen peroxide (H2O2). ROS generation is caused by the simultaneous oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the reduction of NADP+ to NADPH followed by the enzymatic reduction of pyocyanin. The generation of ROS by pyocyanin causes oxidative stress in bacterial, fungal, and mammalian cells which leads to several negative effects including a decrease in NADPH levels, inhibition of essential enzymes, DNA damage, disruption of membrane potential, oxidative damage of the components involved in cell cycle and its regulation.
Pyocyanin is a potent antibacterial agent against both Gram-positive and Gram-negative bacteria, mainly because of its ability to create oxidative stress via ROS production. Meanwhile, pyocyanin inhibits bacterial pathogenesis by hindering biofilm formation which is necessary for bacterial survival and resistance. A study conducted by Laxmi et al. (2016) showed that pyocyanin was able to inhibit biofilm formation in six bacterial species. Pyocyanin is a potential neuroprotective agent due to its inhibitory effect on the enzymatic activity of cellular acetylcholinesterase (AChE). Pyocyanin is a promising treatment for neurodegenerative disorders characterized by elevated levels of the AChE enzyme which hydrolyses the neurotransmitter, acetylcholine. Pyocyanin acts against fungi in a concentration-dependent manner depending on the fungal species. Pyocyanin is a potential treatment for fusariosis, onychomycosis, and keratitis as it demonstrates antifungal activity against Fusarium oxysporum.
Pyocyanin causes a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 μM pyocyanin inducing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been reported in the saliva of the patients infected with P. aeruginosa. Bacterial LPS have been found to have consequential and long-lasting antiapoptotic effects upon neutrophils and are produced in significant concentration by P. aeruginosa. Studies could be conducted to investigate whether P. aeruginosa LPS could inhibit the proapoptotic effect of pyocyanin in co-culture experiments (Fig. 2). After 5 h in culture, LPS (100 ng/ml) significantly retarded constitutive apoptosis (3.8 ± 0.7%) as compared with control (5.9 ± 0.6%, p < 0.05. LPS did not inhibit pyocyanin-induced apoptosis. In three experiments, cells cultured with pyocyanin alone (100 μM) showed no significant difference in apoptosis compared with cells cultured with pyocyanin and LPS at either concentration. Apoptosis was 55.9 ± 5% for pyocyanin-treated populations compared with 60.5 ± 5.3% for cells treated with both pyocyanin and LPS.
Figure 3: Pyocyanin causes neutrophil apoptosis in the presence of LPS. Neutrophils were grown with and without pyocyanin (100 μM) in the absence (▨) or presence (□) of LPS (100 ng/ml), a concentration known to retard constitutive neutrophil apoptosis. After 5 h in culture, LPS inhibited constitutive apoptosis, but was without effect upon pyocyanin-induced cell death. Statistically significant difference (p < 0.05) between means of control and LPS-treated populations.
Pyocyanin is a potential antiapoptotic agent as it has been shown to exhibit cytotoxic effects against different human cancer cell. Pyocyanin has the ability to inhibit the proliferation of cancer cells and decrease cell viability, with its anti-growth and cytotoxic properties. Zhao et al. (2014) experimentally showed the ability of pyocyanin to induce cytotoxicity in HepG2 human hepatoma cells via an increase in DNA damage and cell death. This is mediated by caspase 3 activation and oxidative stress by increased ROS production by glutathione oxidation. Activated caspase 3 is consequential for apoptosis while oxidative stress causes DNA damage e.g apyrimidinic or Apurinic mutations, base pair modifications, and strand breaks. . Pyocyanin’s ability to interfere with cellular functions in normal human cells accelerates the effects of P. aeruginosa infections, especially in immunocompromised individuals, hospital patients, victims of burn wounds and other skin injuries, as well as those suffering from cystic fibrosis (CF).
Pyocyanin induced intestinal microbiota dysbiosis, altering the complex structure of the intestinal barrier. Food and drugs encounter the microbiological barrier in the gut, and Pyocyanin significantly altered the composition of intestinal microbiota. We analyzed the microbiome in various parts of the mouse gut and found increased microbial diversity in the duodenum and ileum, but not in the jejunum and large gut. Lactobacillus decreased in the duodenum, Enterorhabdus decreased in the jejunum, and Bifidobacterium was downregulated in the lower small intestine and large intestine. In contrast, anaerobic bacteria like Ruminococcus, Bacteroides, Prevotella, and Akkermansia were upregulated. Pyocyanin inhibited Lactobacillus growth in vitro and in vivo. The composition and distribution of intestinal microbiota are restricted to specific niches, with aerobic species like Lactobacilli in the upper small intestine and anaerobic bacteria in the large intestine. Pyocyanin may facilitate the translocation of microbiota from the large intestine to the small gut.
Figure 5: Pyocyanin induced duodenal microbiota dysbiosis in mice, with oral administration of 50 mg/kg body weight (n=6). (A) Principal coordinate analysis (PCoA) and (B) comparison of microbial genus counts and α-diversity showed significant changes in gut microbiota.(C) The relative abundance of top 30 genera in the duodenum was altered between CMC-Na (control) and PYO group, with (D) volcano plots and (E) LEfSe analysis revealing differentially abundant taxa. The cladogram displays the taxonomic tree and the histogram represents the LDA scores of bacteria.(F) Growth curve of Pyocyanin inhibited Lactobacillus growth in MHB broth (n=3). Data were shown as mean ± SEM.
In short, Pyocyanin exhibits exceptional anti-bacterial, anti-fungal properties, and anti-biofilm activity; thereby enhancing its applicability medically and clinically. It could be used to prevent and/or eradicate biofilm from the surfaces of medical devices which is a chief source of nosocomial infections. Furthermore, its antioxidant along with its cytotoxic activity against cancer cell lines and minimal impact on normal cell lines, make it a promising contender for use as a substitute for synthetic agents in cancer treatment. However, additional toxicity tests of the extracted pyocyanin are necessary to establish a basis for its regulatory approval.
