Paraoxonase 2 Mediates Metabolic Reprogramming of Murine Tracheal Epithelial Cells in Response to the Quorum Sensing Molecule N‐(3‐oxododecanoyl)‐homoserine Lactone

Abstract

Background The opportunistic pathogen Pseudomonas aeruginosa secretes the quorum sensing (QS) molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) to coordinate gene expression profiles required for the infection of mammalian airways. Paraoxonase 2 (PON2), a ubiquitously-expressed intracellular enzyme cleaves and inactivates C12 via its lactonase activity to disrupt P. aeruginosa QS signaling. C12 is cytotoxic to numerous cell types in a PON2-dependent fashion at supraphysiological concentrations. Our recent work demonstrates that C12 hinders cell proliferation in a PON2-dependent manner at concentrations present in established infections. However, the detailed mechanisms remain unclear. Objective To investigate the mechanism by which C12 hinders cell proliferation at subtoxic concentrations, we monitored metabolic changes in murine tracheal epithelial cells (TECs) isolated from wild type or PON2-deficient mice using liquid chromatography-mass spectrometry (LC-MS), Seahorse real-time cell metabolic analysis, fluorescent microscopy, flow cytometry, and traditional molecular biology approaches. Methods Homozygous PON2-knockout C57BL/6 mice were generated using CRISPR/Cas9. PON2 status was confirmed by sequencing and Western Blot. Primary TECs from wild type or PON2-knockout animals were isolated, cultured, and immortalized using established protocols. Gene expression of tracheal epithelial markers (KRT5, SOX2, TP53) was determined using RT-PCR. Lysates from TECs were analyzed for PON2 activity using LC-MS. Mitochondrial respiration was monitored in TECs with or without PON2 expression treated with vehicle control or C12 using the Seahorse XF Mito Stress Test. Statistical analysis was performed using Student's unpaired t-test. Results Sequencing and western blot analysis indicate functional PON2 expression in wild type, but not PON2-KO, mice. RT-PCR analysis shows similar expression of tracheal epithelial markers in both WT and PON2-KO TECs post-immortalization. LC-MS analysis reveals PON2 enzymatic activity in WT TECs, but not PON2-KO TECs. Treatment with C12 hinders mitochondrial oxygen consumption, promotes extracellular acidification, and disrupts mitochondrial structure in TECs with PON2 expression. Conclusions These findings indicate that exposure to subtoxic concentrations of C12 alters components of cellular metabolism in a PON2-dependent manner.