Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium
Haider H. Dar, … , Hülya Bayır, Valerian E. Kagan
Haider H. Dar, … , Hülya Bayır, Valerian E. Kagan
Published September 10, 2018
Citation Information: J Clin Invest. 2018;128(10):4639-4653. https://doi.org/10.1172/JCI99490.
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Research Article Cell biology Infectious disease

Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium

Abstract

Ferroptosis is a death program executed via selective oxidation of arachidonic acid–phosphatidylethanolamines (AA-PE) by 15-lipoxygenases. In mammalian cells and tissues, ferroptosis has been pathogenically associated with brain, kidney, and liver injury/diseases. We discovered that a prokaryotic bacterium, Pseudomonas aeruginosa, that does not contain AA-PE can express lipoxygenase (pLoxA), oxidize host AA-PE to 15-hydroperoxy-AA-PE (15-HOO-AA-PE), and trigger ferroptosis in human bronchial epithelial cells. Induction of ferroptosis by clinical P. aeruginosa isolates from patients with persistent lower respiratory tract infections was dependent on the level and enzymatic activity of pLoxA. Redox phospholipidomics revealed elevated levels of oxidized AA-PE in airway tissues from patients with cystic fibrosis (CF) but not with emphysema or CF without P. aeruginosa. We believe that the evolutionarily conserved mechanism of pLoxA-driven ferroptosis may represent a potential therapeutic target against P. aeruginosa–associated diseases such as CF and persistent lower respiratory tract infections.

Authors

Haider H. Dar, Yulia Y. Tyurina, Karolina Mikulska-Ruminska, Indira Shrivastava, Hsiu-Chi Ting, Vladimir A. Tyurin, James Krieger, Claudette M. St. Croix, Simon Watkins, Erkan Bayir, Gaowei Mao, Catherine R. Armbruster, Alexandr Kapralov, Hong Wang, Matthew R. Parsek, Tamil S. Anthonymuthu, Abiola F. Ogunsola, Becca A. Flitter, Cody J. Freedman, Jordan R. Gaston, Theodore R. Holman, Joseph M. Pilewski, Joel S. Greenberger, Rama K. Mallampalli, Yohei Doi, Janet S. Lee, Ivet Bahar, Jennifer M. Bomberger, Hülya Bayır, Valerian E. Kagan

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Figure 3

P. aeruginosa–induced ferroptosis is affected by manipulations of PLs in HBE cells.

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P. aeruginosa–induced ferroptosis is affected by manipulations of PLs i...
(A) Exogenous AA enhances P. aeruginosa–induced ferroptosis. HBE cells were treated with WT, ΔwspF, or loxA::Tn supernatant (Sup.) alone or in combination with AA (2.5 μM) for 20 hours at 37°C. Cell death was estimated as mentioned above. Data are presented as mean ± SD, *P < 0.05 vs. control (untreated), #P < 0.05 vs. corresponding supernatant only, †P < 0.05 vs. corresponding AA treatment; n = 3. (B) HBE cells were transfected with scrambled siRNA (si-NC) or siRNA against ACSL4 or LPCAT3. Transfected cells were treated with ΔwspF supernatants with or without ferrostatin-1 (0.2 μM) for 20 hours at 37°C before estimating cell death. Data are presented as mean ± SD; *P < 0.05 vs. si-NC ΔwspF; n = 3. (C) Ferrostatin-1 inhibits P. aeruginosa–induced biofilm formation on HBE cells. Polarized HBE cells were incubated with ΔwspF P. aeruginosa (MOI of 25) for 1 hour. After washing unattached bacteria, HBE cells were cultured in the absence or presence of ferrostatin-1 (0.2 and 1.0 μM) for 5 hours. Biofilms were removed and lysed with Triton X-100 (0.1%), and the number of bacteria in each sample was determined by CFU assay. Data are presented as mean ± SD, *P < 0.05 vs. ΔwspF with no FER; n = 3. (D) Treatment of HBE cells with ΔwspF supernatant decreases the content of GPX4. Cells were treated with ΔwspF supernatant and incubated for 20 hours at 37°C. Samples were collected and processed for Western blotting to determine the levels of GPX4. RSL3, a covalent GPX4 inhibitor, was used as a positive control. For quantification, the band intensity of GPX4 protein was normalized to respective band intensity of actin. Inset: typical Western blot (of 3 performed). Data are presented as mean ± SD, *P < 0.05 vs. control (untreated) HBE cells; n = 3. (E) Exogenous 15-HOO-AA-PE elevated WT but not loxA-deficient loxA::Tn supernatant–induced cell death. HBE cells pretreated with RSL3 (20 nM for 4 hours) were incubated with PE-OOH alone or with supernatant from loxA::Tn or WT in the presence or absence of ferrostatin-1 (0.2 μM). Data are presented as mean ± SD, *P < 0.05 vs. control (untreated) HBE cells, #P < 0.05 vs. FER; n = 3. One-way ANOVA.