PON2 mediates mitochondrial dysfunction in tracheal epithelial cells in response to a quorum sensing molecule N-(-3-oxododecanoyl)-l-homoserine lactone

Abstract

The opportunistic bacterium Pseudomonas aeruginosa secretes the quorum-sensing molecule N-(3-oxododecanoyl)-l-homoserine lactone (C12) to coordinate gene expression profiles favorable for infection. Recent studies have demonstrated that high concentrations of C12 impair many aspects of host cell physiology, including mitochondrial function and cell viability. The cytotoxic effects of C12 are mediated by the lactonase enzyme, Paraoxonase 2 (PON2), which hydrolyzes C12 to a reactive metabolite. However, the influence of C12 on host cell physiology at concentrations observed in patients infected with P. aeruginosa is largely unknown. Since the primary site of P. aeruginosa infections is the mammalian airway, we sought to investigate how PON2 modulates the effects of C12 at subtoxic concentrations using immortalized murine tracheal epithelial cells (TECs) isolated from wild type (WT) or PON2-knockout (PON2-KO) mice. Our data reveal that C12 at subtoxic concentrations disrupts mitochondrial bioenergetics to hinder cellular proliferation in TECs expressing PON2. Subtoxic concentrations of C12 disrupt normal mitochondrial network morphology in a PON2-dependent manner without affecting mitochondrial membrane potential. In contrast, higher concentrations of C12 depolarize mitochondrial membrane potential and subsequently trigger caspase signaling and apoptotic cell death. These findings demonstrate that different concentrations of C12 impact distinct aspects of host airway epithelial cell physiology through PON2 activity in mitochondria.

Paraoxonase 2 (PON2) plays a limited role in murine lung tumorigenesis

“…While control-shRNA LLC cells exhibited a concentration-dependent increase in cell death and caspase-3/7 activation upon C12 exposure, cells lacking PON2 expression were resistant to the cytotoxicity of C12 at all concentrations (Fig. 1 B,C), in agreement with earlier studies of various cell types 16 – 19 . Next, we examined cellular proliferation in LLCs with or without PON2 expression.…”

“…1 A). To further validate the reduction of PON2 expression, we conducted cell viability and caspase-3/7 activity assays in control- and PON2-shRNA LLC cells treated with increasing concentrations of the bacterial quorum-sensing molecule N -(3-oxododecanoyl)-l-homoserine lactone (C12), a specific inducer of PON2-mediated apoptosis 16 – 19 . While control-shRNA LLC cells exhibited a concentration-dependent increase in cell death and caspase-3/7 activation upon C12 exposure, cells lacking PON2 expression were resistant to the cytotoxicity of C12 at all concentrations (Fig.…”

“…Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) and incubated with indicated primary or secondary antibodies in blotting buffer containing 1X phosphate-buffered saline (PBS), 0.2% Tween-20, and 10% (w/v) non-fat dry milk (Thermo Fisher). The enhanced chemiluminescence detection system (Thermo Fisher) was used to detect proteins as previously described 19 .…”

“…Following treatment with the indicated agents, cells were treated with trypsin and collected with addition of final concentration of 1 µg/mL PI. Cell viability was determined via PI exclusion using flow cytometry as described earlier 19 . The percentage of cell death was calculated as100 minus the value of cell viability measurement.…”

“…Eight-week-old male and female Kras LSL-G12D (B6.129S4-Kras tm4Tyj /J; 008179) mice were purchased from Jackson laboratories (Bar Harbor, ME). PON2-KO mice were described in our earlier studies 19 . Kras LSL-G12D mice were selectively bred with PON2-KO mice to generate PON2-heterozygous mice harboring the mutant Kras LSL-G12D allele.…”