Pyocyanin Induces Mitochondrial Dysfunction In The Biofilm Infected Cutaneous Wound Through Upregulation Of Mir-146a
Karamjeet Kaur, Mithun Sinha, Sashwati Roy, Chandan K. Sen.
Indiana University, Indianapolis, IN, USA.
BACKGROUND- Pyocyanin is a redox active secondary metabolite secreted by Pseudomonas aeruginosa. In the biofilm infected wounded skin, it penetrates cell membrane and disrupts intracellular redox homeostasis via mitochondrial dysfunctioning. This work shows that wound-site pyocyanin causes mitochondrial dysfunction by upregulating a mito-miR, miR-146a.
METHODS- Human keratinocytes were treated with 10µM pyocyanin for 5d. Such levels have been detected in patient wound fluid. miR-146a levels were determined via qRT-PCR. Oxygen Consumption Rate (OCR), respiration, proton leak and ATP production was measured using Sea Horse Analyzer XFe96. Mitochondrial untranslated protein response (UPRmt) was analyzed.
RESULTS- QRT-PCR data showed that pyocyanin (10µM) treatment caused two-fold induction of miR-146a in keratinocytes (n=4, p<0.05). Upregulated miR-146a targets mitochondrial transcripts such as SOD2 causing mitochondrial dysfunction. In order to study mechanism underlying such mitochondrial dysfunctioning, pyocyanin (10µM) treated cells and miR-146a mimic transfected cells were analyzed on Sea Horse Analyzer XFe96. OCR (pmoles/ min) was significantly reduced in both the conditions (pyocyanin treated and miR-146a transfected cells n=4, p<0.05). miR-146a compromised cellular respiration (non-mitochondrial, basal and maximal) in keratinocytes. Under the same conditions, proton leak was also observed. Such leak may be viewed as sequelae of distorted or leaky mito-membrane. ATP production dropped from 40 pmoles/ min to 20 pmoles/ min in the pyocyanin treated/ miR-146a transfected cells. Pyocyanin treatment also disrupted mitochondrial proteostasis which stimulated UPRmt (n=4, p<0.05). Elevated UPRmt is known to upregulate mitochondrial folding capacity in response to stress. Such response is aimed at circumventing deleterious protein aggregation. In cells with elevated UPRmt, transcription factors NFκB and p53 were upregulated.
CONCLUSION- Presence of pyocyanin in the wounded skin disrupts mitochondrial bioenergetics which complicates wound healing. The disrupted mitochondrial bioenergetics is contributed by miR-146a induced by pyocyanin. Elevated miR-146a, thus, represents a key mechanism in this paradigm.
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