Biofilm Infection Poses Risk Of Oxidative Stress Via Redox Cycling Of Secretory Pyocyanin
Karamjeet Kaur, James Boslett, Craig Hemann, Jay L. Zweier, Chandan K. Sen.
Comprehensive Wound Center, The Ohio State University, Columbus, OH, USA.
Background- While at low concentrations reactive oxygen species (ROS) are known to drive cell signaling towards wound healing, excessive ROS such as during chronic inflammation and diabetes stall wound repair. This work shows that biofilm infection may add to the burden of oxidative stress at the wound site by redox cycling of pyocyanin. Pyocyanin (5-methyl-1-hydroxyphenazine) is a secretory product of Pseudomonas aeruginosa. Methods- Engineered human skin was treated with 10µM pyocyanin for 3d. Electron Paramagnetic Resonance (EPR) studies of the treated skin was conducted. The change in reducing equivalents (NADH, NADPH and GSH) pool was also determined in 10µM pyocyanin treated human HaCaT keratinocytes. Results- EPR spectrum of 10µM pyocyanin treated engineered human skin showed the presence of reduced pyocyanin radical which following UV exposure for 30 minutes generated superoxide and hydroxyl radicals. Thus, exposure to direct sunlight can worsen the already damaged biofilm infected wound site. A decline in NADH, NADPH and GSH levels was observed in 10µM pyocyanin treated keratinocytes as compared to untreated cells. Depletion of reducing equivalents was caused by the transfer of electrons from reducing equivalents to pyocyanin which helps bacteria to modulate their intracellular redox state. Modulation of intracellular redox state is crucial for survival of bacteria at high cell densities when there is electron-acceptor limitation, so that they can form thick biofilm. As this is achieved, there is more pyocyanin production, more ROS generation, continuous depletion of reducing equivalents pool and increased oxidative stress which does not allow the biofilm infected wound site to recover. Conclusion- Pyocyanin is cytotoxic to human cells due to its redox-active nature. Such toxicity markedly magnifies upon UV exposure. Pyocyanin oxidizes cellular reducing equivalents, induces oxidative stress and is therefore likely to complicate wound healing.
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