Pyocyanin effects on respiratory epithelium: relevance in Pseudomonas aeruginosa airway infections

Abstract

Pseudomonas aeruginosa (PA) uses several virulence factors to establish chronic respiratory infections in bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis (CF) patients. One of its toxins, pyocyanin (PYO), is a redox-active pigment that is required for full virulence in animal models and has been detected in patients' airway secretions. PYO promotes virulence by interfering with several cellular functions in host cells including electron transport, cellular respiration, energy metabolism, gene expression, and innate immune mechanisms. This review summarizes recent advances in PYO biology with special attention to current views on its role in human airway infections and on its interactions with the first line of our airway defense, the respiratory epithelium.

Figure 1. Pyocyanin (PYO) is a redox-active exotoxin of Pseudomonas aeruginosa

(a) Depicted are chemical structures of chorismic acid, the starting point for constructing the phenazine aromatic frame, and two precursors involved in the finals steps of PYO biosynthesis. (b) PYO is produced by stationary phase cultures of P. aeruginosa PAO1. The PhzM-deficient P. aeruginosa PA14 strain fails to turn the medium dark and produce pyocyanin. PYO can be purified from stationary phase Pseudomonas cultures by repeated chloroform–distilled water extraction cycles. (c) The absorption spectrum of PYO is pH-dependent. At low pH values PYO solutions are red, whereas at higher pH values it turns blue. (d) Well-oxygenated stationary phase cultures of PA14 are dark green due to high oxidized PYO concentrations (left). In standing cultures the medium loses its dark color as reduced PYO accumulates over time (3–20 min). Upon reoxygenation oxidized PYO turns the culture medium green again (right, 20 min). (e) Standing cultures (anaerobic conditions) show a characteristic green-yellow gradient near the air-medium surface because PYO shuffles electrons between oxygen-poor parts of the culture and the air surface.

Figure 2. Pyocyanin (PYO) exposes host cells to oxidative stress

(a) PYO directly oxidizes reduced NAD(P)H in the host cell cytoplasm and donates accepted electrons to oxygen molecules to produce superoxide anions and downstream reactive oxygen species (ROS) (GSH/GSSG: reduced:oxidized glutathione ratio). In a cell-free system NADPH consumption (b) and superoxide production (c) is enhanced by increasing concentrations (0–100 μM) of PYO. NADPH consumption detected by 260 nm absorbance changes; superoxide release detected by Diogenes luminescence. (d) Oxidative stress in bronchial epithelial (H292) cells exposed to PYO (20 μM, 60 min) is visualized by increased intracellular fluorescence of the ROS-sensitive fluorescence dye dihydrorhodamine 123.

Figure 3. Mechanistic model of the proinflammatory action of pyocyanin (PYO) in the respiratory epithelium

PYO produced by Pseudomonas aeruginosa enters the cytosol of airway epithelial cells and produces reactive oxygen species (ROS) by oxidizing its intracellular NADPH pool. Primary effects resulting from this are diminished reduced glutathione (GSH) and ATP levels, increased cytosolic redox potential (E_redox) and intracellular oxidative stress. Reduced GSH levels affect energy metabolism of the cells and inhibit ciliary beat frequency, V-ATPase activity and CFTR channel functions. Reduced NADPH levels inhibit antibacterial functions (bacterial killing) and PYO inactivation (mediated by peroxidases) due to reduced activity of epithelial NADPH oxidases, Duox1 and Duox2. Long-term effects of PYO manifest in transcriptional changes. PYO inhibits gene expression of the antioxidant and antimicrobial genes catalase and Duox. Several genes are induced in response to PYO including: oxidative stress-responsive genes, mucins, inflammatory cytokines and chemokines. The inflammatory changes evoked by PYO can be grouped into two major effects. First, PYO induces several genes mediating neutrophil recruitment to the airways. Second, PYO also induces mucin production in the airway epithelium mainly through activation of the EGFR signaling pathway. Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; CXCL, chemokine (C-X-C motif) ligand; Duox, dual oxidase; E_redox, redox potential; EGFR, epidermal growth factor receptor; IL, interleukin; LPO, lactoperoxidase; MPO, myeloperoxidase; GSH, reduced glutathione; GSSG, oxidized glutathione.