Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury. B E Britigan, … , M L McCormick, C D Cox J Clin Invest. 1992;90(6):2187-2196. https://doi.org/10.1172/JCI116104. Research Article Pyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the […] Find the latest version: https://jci.me/116104/pdf Interaction of the Pseudomonas aeruginosa Secretory Products Pyocyanin and Pyochelin Generates Hydroxyl Radical and Causes Synergistic Damage to Endothelial Cells Implications for Pseudomonas-associated Tissue Injury Bradley E. Bntigan, * * Tedmund L. Roeder, George T. Rasmussen,t D. Michael Shasby, * * Michael L. McCormick,t and Charles D. Cox" Research Service and Department of Internal Medicine, VA Medical Center, Iowa City, Iowa 52246; and Departments of tInternal Medicine and OMicrobiology, University ofIowa College ofMedicine, Iowa City, Iowa 52242 Abstract Introduction Pyocyanin, a secretory product of Pseudomonas aeruginosa, Nosocomial pneumonia remains a major cause of morbidity has the capacity to undergo redox cycling under aerobic condi- and mortality in the United States. In most clinical series Pseu- tions with resulting generation of superoxide and hydrogen per- domonas aeruginosa ranks among the top three Gram-nega- oxide. By using spin trapping techniques in conjunction with tive organisms causing such infections ( 1 ). Pseudomonas pneu- electron paramagnetic resonance spectrometry (EPR), super- monia is particularly severe with a mortality approaching 70% oxide was detected during the aerobic reduction of pyocyanin by even with appropriate antibiotic therapy ( 1, 2). P. aeruginosa NADH or porcine endothelial cells. No evidence of hydroxyl also plays a major role in the chronic progressive pulmonary radical formation was detected. Chromium oxalate eliminated dysfunction in patients with cystic fibrosis, which is manifested the EPR spectrum of the superoxide-derived spin adduct re- as progressive bronchiectasis and hemoptysis (3) and which sulting from endothelial cell exposure to pyocyanin, suggesting currently accounts for > 90% of deaths in these patients (4). It superoxide formation close to the endothelial cell plasma mem- is unclear why Pseudomonas lung infections carry such a high brane. We have previously reported that iron bound to the P. fatality rate. Most work has focused on the destructive nature aeruginosa siderophore pyochelin (ferripyochelin) catalyzes of Pseudomonas-derived proteases and other toxins (2, 4). the formation of hydroxyl free radical from superoxide and hy- However, alternative mechanisms may be involved. drogen peroxide via the Haber-Weiss reaction. In the present Many P. aeruginosa strains secrete pyocyanin, a phenazine study, spin trap evidence of hydroxyl radical formation was derivative (5-methyl- 1-hydroxyphenazine), which is toxic for detected when NADH and pyocyanin were allowed to react in eukaryotic and prokaryotic cells (5-7). Wilson et al. (8) re- the presence of ferripyochelin. Similarly, endothelial cell expo- ported that pyocyanin, at concentrations present in the sputum sure to pyocyanin and ferripyochelin also resulted in hydroxyl sol of cystic fibrosis patients, induced ciliary dysfunction in radical production which appeared to occur in close proximity human nasal epithelial cells (9, 10). Like paraquat, mena- to the cell surface. As assessed by 51Cr release, endothelial cells dione, and other xenobiotics, pyocyanin can redox cycle in the which were treated with pyocyanin or ferripyochelin alone dem- presence of various reducing agents and oxygen with resultant onstrated minimal injury. However, endothelial cell exposure formation of superoxide (O-°) and hydrogen peroxide (H202) to the combination of pyochelin and pyocyanin resulted in 55% (5). Pyocyanin-induced free radical production appears to be specific 51Cr release. Injury was not observed with the substitu- responsible for much of its microbicidal activity (5), but its tion of iron-free pyochelin and was diminished by the presence role in P. aeruginosa-associated tissue injury is less clear. of catalase or dimethyl thiourea. These data suggest the possi- Superoxide and H202 are only moderately reactive oxi- bility that the P. aeruginosa secretory products pyocyanin and dants. In the presence of an exogenous iron catalyst, *0° and pyochelin may act synergistically via the generation of hy- H202 react to form hydroxyl radical via the Haber-Weiss reac- droxyl radical to damage local tissues at sites of pseudomonas tion ( 11). This extremely reactive and toxic oxidant has been infection. (J. Clin. Invest. 1992. 90:2187-2196.) Key words: incriminated as the actual mediator of several forms of cell free radical * iron * pyocyanin * siderophore * spin trap * super- injury associated with O / H202 exposure ( 12). In vivo, extra- oxide cellular iron is tightly complexed to host iron-binding proteins such as transferrin and lactoferrin. These iron chelates cannot participate in the Haber-Weiss reaction ( 13-15), thereby pro- tecting tissue from the potentially damaging effects of unto- This work was presented in part on 2 October 1991 at the Interscience ward hydroxyl radical formation (16). Although some intra- Conference of Antimicrobial Agents and Chemotherapy in Chicago, IL. cellular iron chelates such as ferritin and haemosiderin have Address reprint requests to Dr. Britigan, Department of Internal been reported to be capable of acting as hydroxyl radical cata- Medicine, SW54, GH, University of Iowa College of Medicine, Iowa lysts ( 17, 18), the presence of intracellular antioxidants such as City, IA 52246. the superoxide dismutases and catalase limit the access of O2 Receivedfor publication 22 April 1992 and in revisedform 22 July and H202 to them. This suggests that pathologic in vivo hy- 1992. droxyl radical generation may sometimes depend on the pres- ence of nonphysiologic iron chelates. The Journal of Clinical Investigation, Inc. Like many microorganisms, P. aeruginosa secretes iron- Volume 90, December 1992, 2187-2196 binding compounds (siderophores) which allow the organism Pyocyanin and Pyochelin Generate Hydroxyl Radical 2187 to compete with host iron-binding proteins for iron ( 19, 20). Microtiter plates. Porcine pulmonary artery endothelial cells were Recently, we found that iron bound to the P. aeruginosa sider- plated at a concentration of 5 x 104 cells per well in 24-well tissue ophore pyochelin was capable of catalyzing the formation of culture plates (Costar, Cambridge, MA) in 0.5 ml medium 199 with hydroxyl radical from a continuous flux of 0 /H202 gener- 10% serum, 2x basal medium amino acids, basal minimal essential 10 U/ml penicillin/streptomycin or a enzymatic system vitamins, 2 mM L-glutamine, ated by stimulated neutrophils cell-free (Gibco Laboratories, Grand Island, NY). The plates were then placed (21 ). These data suggested that ferripyochelin might contrib- in culture at 370C, 5% CO2 for 4-5 d. ute to Pseudomonas-mediated tissue injury via the generation of cytotoxic hydroxyl radical. Spin trapping Given: the evidence that Pseudomonas-derived secretory Desired reaction mixtures (0.5 ml) were assembled in 12 X 75-mm products could lead to generation of oxygen-centered free radi- glass tubes and transferred to a quartz electron paramagnetic resonance cals; the tendency of this microorganism to invade vascular (EPR) flat cell which was in turn placed into the cavity of the EPR structures (22); the substantial evidence for the susceptibility spectrometer (model E 104A, Varian Associates, Inc., Palo Alto, CA; or of pulmonary endothelial cells to oxidant injury; and a role for model ESP300, Bruker, Karlsruhe, FRG). Spin trapping systems con- such endothelial cell injury in the pathogenesis of several types sisted of 0. 1 mM diethylenetriaminepentaacetic acid (DTPA) and the following: 100 mM 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) with