Role of Pyocyanin and Extracellular DNA in Facilitating Pseudomonas aeruginosa Biofilm Formation

Pseudomonas aeruginosa is an opportunistic Gram‐negative bacterium that is primarily responsible for infections related to cystic fibrosis (CF) airways, burn wounds, urinary tract infections, surgery‐associated infections, and HIV‐related illness. Pyocyanin and extracellular DNA (eDNA) are the major factors dictating the progression of biofilm formation and infection. Pyocyanin is a potent virulence factor causing cell death in infected CF patients and is associated with high mortality. eDNA is a key player in P. aeruginosa biofilm formation and is also responsible for the high viscosity of CF sputum that blocks the respiratory airway passages. In this chapter, we summarize our recent findings on the role of pyocyanin in facilitating P. aeruginosa biofilm formation. Pyocyanin promotes eDNA release in P. aeruginosa by inducing cell lysis mediated via hydrogen peroxide (H2O2) production. Pyocyanin intercalates with the nitrogenous bases of DNA and creates structural perturbation on the double‐helix structure. Pyocyanin‐eDNA binding significantly influences P. aeruginosa cell surface hydrophobicity and influences the physicochemical interactions facilitating bacterial cell‐to‐cell interaction (aggregation) and ultimately facilitates robust biofilm formation. A pyocyanin knockout (ΔphzA‐G) mutant is shown to have significantly reduced eDNA release and biofilm formation in comparison to its wild‐type. To this end, we discover that antioxidant glutathione directly binds to pyocyanin and modulates pyocyanin structure and function, thus inhibiting pyocyanin‐eDNA binding and consequently hampering biofilm development

Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

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

Glutathione enhances antibiotic efficiency and effectiveness of DNase I in disrupting Pseudomonas aeruginosa biofilms while also inhibiting pyocyanin activity, thus facilitating restoration of cell enzymatic activity, Confluence and Viability

© 2017 Das, Simone, Ibugo, Witting, Manefield and Manos. Pyocyanin secreted by Pseudomonas aeruginosa is a virulence factor that damages epithelial cells during infection through the action of reactive oxygen species, however, little is known about its direct effect on biofilms. We demonstrated that pyocyanin-producing P. aeruginosa strains (PA14WT, DKN370, AES-1R, and AES-2) formed robust biofilms in contrast to the poorly formed biofilms of the pyocyanin mutant PA14ΔphzA-G and the low pyocyanin producer AES-1M. Addition of DNase I and reduced glutathione (GSH) significantly reduced biofilm biomass of pyocyanin-producing strains (P < 0.05) compared to non-pyocyanin producers. Subsequently we showed that a combined treatment comprising: GSH + DNase I + antibiotic, disrupted and reduced biofilm biomass up to 90% in cystic fibrosis isolates AES-1R, AES-2, LESB58, and LES431 and promoted lung epithelial cell (A549) recovery and growth. We also showed that exogenously added GSH restored A549 epithelial cell glutathione reductase activity in the presence of pyocyanin through recycling of GSSG to GSH and consequently increased total intracellular GSH levels, inhibiting oxidative stress, and facilitating cell growth and confluence. These outcomes indicate that GSH has multiple roles in facilitating a return to normal epithelial cell growth after insult by pyocyanin. With increased antibiotic resistance in many bacterial species, there is an urgency to establish novel antimicrobial agents. GSH is able to rapidly and comprehensively destroy P. aeruginosa associated biofilms while at a same time assisting in the recovery of host cells and re-growth of damaged tissue