Pseudomonas aeruginosa Induced Host Epithelial Cell Mitochondrial Dysfunction

Abstract

The pathogenicity of P. aeruginosa is dependent on quorum sensing (QS), an inter-bacterial communication system that can also modulate host biology. The innate immune function of the lung mucosal barrier is dependent on proper mitochondrial function. The purpose of this study was to define the mechanism by which bacterial factors modulate host lung epithelial cell mitochondrial function and to investigate novel therapies that ameliorate this effect. 3-oxo-C12-HSL disrupts mitochondrial morphology, attenuates mitochondrial bioenergetics, and induces mitochondrial DNA oxidative injury. Mechanistically, we show that 3-oxo-C12-HSL attenuates expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, antioxidant defense, and cellular respiration, and its downstream effectors in both BEAS-2B and primary lung epithelial cells. Overexpression of PGC-1α attenuates the inhibition in cellular respiration caused by 3-oxo-C12-HSL. Pharmacologic activation of PGC-1α restores barrier integrity in cells treated with 3-oxo-C12-HSL. These data demonstrate that the P. aeruginosa QS molecule, 3-oxo-C12-HSL, alters mitochondrial pathways critical for lung mucosal immunity. Genetic and pharmacologic strategies that activate the PGC-1α pathway enhance host epithelial cell mitochondrial function and improve the epithelial innate response to P. aeruginosa. Therapies that rescue PGC-1α function may provide a complementary approach in the treatment of P. aeruginosa infection.

Figure 1

P. aeruginosa QS molecules disrupt mitochondrial morphology in bronchial epithelial cells. BEAS-2B cells were treated with vehicle control (DMSO), 100 μM 3-oxo-C12-HSL (C12-HSL), or infected with PAO1 (MOI 20) for 6 hours. Cells were then fixed and processed for electron microscopy (EM) analysis. (A) representative high-powered field (hpf) images of treatment groups, 15,000x magnification, scale 0.2 μm. (B) number of mitochondria per hpf. (C) Total mitochondrial area per hpf. (D) Mitochondrial length to width ratio. 3-oxo-C12-HSL and PAO1 disrupt mitochondrial morphologic parameters. Results are mean ± SEM. *P < 0.05, **p < 0.01, ****p < 0.0001 all vs. control. One-way ANOVA with Tukey’s multiple comparisons test used for statistical analysis.

Figure 2

P. aeruginosa QS molecules disrupt bronchial epithelial cell bioenergetics and metabolic potential. BEAS-2B cells were treated with vehicle control (DMSO) or 100 μM 3-oxo-C12-HSL for 6 hours. Cells were then analyzed using Seahorse Cell Energy Phenotype assay. This allows for the real time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are representative of mitochondrial respiration and glycolysis respectively, at rest and after induction of a bioenergetic stress caused by treatment with the ATP synthase inhibitor, oligomycin, and the uncoupling agent, FCCP. (A) Plot of baseline and stressed conditions in control and 3-oxo-C12-HSL-treated cells. 3-oxo-C12-HSL-treated cells are more quiescent at baseline and have less metabolic potential under stressed conditions. (B) OCR measurements at baseline and stressed conditions. 3-oxo-C12-HSL decreases basal and stressed OCR as compared to control. (C) ECAR measurements at baseline and stressed conditions. 3-oxo-C12-HSL decreases basal and stressed ECAR as compared to control. Results are mean ± SEM. *p < 0.05, n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test used for statistical analysis.

Figure 3

3-oxo-C12-HSL attenuates mitochondrial respiration in bronchial epithelial cells. (A) BEAS-2B cells were treated with 100 μM 3-oxo-C12-HSL for 6 hours and then the amount of ATP normalized to protein was measured in cellular lysates. 3-oxo-C12-HSL significantly reduced the level of ATP present in cells. (B–F) BEAS-2B cells were treated with 50, 100, or 200 μM 3-oxo-C12-HSL for 6 hours and then analyzed using the Seahorse XF Mito Stress Test assay. (B) OCR was measured real time at baseline and then in response to a series of injections to interrogate various aspects of the electron transport chain. 3-oxo-C12-HSL significantly attenuated basal respiration (C) maximal respiration (D) spare respiratory capacity (E) and ATP-linked respiration (F). Results are mean ± SEM. For A, * p < 0.05 by unpaired two-tailed t test, n = 3 independent experiments. For C-F * p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test, n = 6 independent experiments.

Figure 4

P. aeruginosa infection and the bacterial QS molecule attenuates expression of PGC-1α and TFAM and reduces mitochondrial biogenesis in bronchial epithelial cells. (A–C,E–G,I–J) BEAS-2B cells were treated with 100 μM 3-oxo-C12-HSL or infected with PAO1 (MOI 1) for 16 hours. Relative mRNA (A,E,I) and protein expression (B,F for representative western blots, (C,G for normalized densitometry) of PGC-1α (B,C) and TFAM (F,G) were measured. The full-length blots are presented in Supplementary Figs 1 and 2. Both PAO1 and 3-oxo-C12-HSL significantly attenuated expression of PGC-1α and TFAM in BEAS-2B cells. (D,H,K) Human primary bronchial epithelial cells (NhBEs) grown at an air-liquid interface were also treated with 100 μM 3-oxo-C12-HSL or infected with PAO1 (MOI 1) for 6 and 18 hours and relative mRNA expression for PGC-1α (D) and TFAM (H) were measured. PAO1 attenuated expression of PGC-1α and TFAM at both 6 and 18 hours. 3-oxo-C12-HSL attenuated expression of PGC-1α and TFAM at 18 hours. (I) In BEAS-2B cells, 3-oxo-C12-HSL reduced relative mRNA expression of the mitochondrial marker VDAC1. In BEAS-2B cells (J) and NhBEs (K) 3-oxo-C12-HSL reduced relative mtDNA content normalized to nuclear DNA. Results are mean ± SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test, n = 6–8 independent experiments (A,C,E,G,I) n = 3 independent experiments (D,H). For (I,J,K) results are mean ± SEM.*p < 0.05 by unpaired t test, n = 3 independent experiments.

Figure 5

P. aeruginosa QS molecule induces ROS generation and oxidative mtDNA injury in bronchial epithelial cells. (A–C) BEAS-2B cells were treated with 3-oxo-C12-HSL (10 μM, 100 μM), or PAO1 (MOI 20), or a positive control (rotenone and antimycin A, R/A) for 6 hours. MitoSOX fluorescence was measured using flow cytometry. (A) Mean fluorescence intensity is displayed in a histogram format. Cells that stained positive for MitoSOX fluorescence (labeled Positive Cells) were quantified and compared between groups (B). 100 μM 3-oxo-C12-HSL resulted in a significant increase in ROS generation. (C,D) The ratio of a 79 bp mtDNA fragment to 230 bp mtDNA fragment was used to quantify mtDNA oxidative damage in BEAS-2B cells (C) and primary NhBEs (D) treated with 100 μM 3-oxo-C12-HSL or PAO1 for 6 hours. 100 μM 3-oxo-C12-HSL produced significant mtDNA oxidative injury in both BEAS-2B and NhBEs. A-C, Results are mean ± SEM. *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n = 3 independent experiments. (D) Results are mean ± SEM. *p < 0.05, unpaired t test, n = 3 independent experiments

Figure 6

P. aeruginosa QS molecule triggers the apoptosis pathway. (A) BEAS-2B cells were treated with 100 μM 3-oxo-C12-HSL for 6 hours. The intracellular quantity of cytochrome c normalized to protein was quantified in the two groups. (B) BEAS-2B cells were treated with 100 μM 3-oxo-C12-HSL for 3, 6, and 12 hours and then protein was isolated and used for immunoblotting for the cleaved and full-length forms caspase-8 and PARP relative to the endogenous control, GAPDH. Immunoblots shown are representative of three independent experiments. (C) BEAS-2B cells were treated with100 μM 3-oxo-C12-HSL for 1, 3, and 6 hours and caspase-3 activity was measured. 3-oxo-C12-HSL induced cytochrome c release, caspase-8 cleavage, caspase-3 activation, and PARP cleavage. (A,C) Results are mean ± SEM. *p < 0.05 by unpaired t test (A) or one-way ANOVA with Tukey’s multiple comparisons test (C) n = 3 independent experiments.

Figure 7

Overexpression of PGC-1α partially rescues 3-oxo-C12-HSL-induced impairment in mitochondrial biogenesis and cellular respiration. BEAS-2B cells were transduced with adenovirus (MOI 25) expressing GFP (AdGFP) or PGC-1α (AdPGC-1α) for 48 hours (A,B) or 72 hours (C–H) prior to treatment with 100 μM 3-oxo-C12-HSL. QPCR for PGC-1α (A) and TFAM (B) and western blot representative of three independent experiments for PGC-1α and TFAM (C) confirms that PGC-1α overexpression prevents the attenuation in the expression of PGC-1α and TFAM caused by 3-oxo-C12-HSL. Full length blots are presented in Supplementary Fig. 3D–H. Seahorse XF Mito Stress Test analysis demonstrates that in cells treated with 3-oxo-C12-HSL, PGC-1α overexpression significantly increased basal respiration (E) maximal respiration (F) spare respiratory capacity, and ATP-linked respiration (G). Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test, n = 4 (A–C) or 6 (D–G) independent experiments.

Figure 8

Pre-treatment with metformin or resveratrol partially rescues impairment in mitochondrial biogenesis and restores epithelial barrier integrity following 3-oxo-C12-HSL treatment and prevents bacterial transmigration. (A,B) BEAS-2B cells were pre-treated with either metformin 1 mM (A) or resveratrol 20 μM (B) for 24 hours prior to 12-hour treatment with 100 μM 3-oxo-C12-HSL and relative mRNA expression of TFAM was measured by QPCR. The attenuation in TFAM expression caused by 3-oxo-C12-HSL was blocked by prior treatment with metformin or resveratrol. (C) Calu-3 cells grown on transwell inserts were pretreated with either vehicle control, metformin 1 mM, or resveratrol 20 μM overnight and then inoculated with PAO1 (MOI 1) in the apical media for 6 hours. The basolateral media was collected, serially diluted, and cultured to quantify cfu. Resveratrol or metformin pretreatment significantly decreased the degree of bacterial transmigration. (D) BEAS-2B cells pretreated with either metformin1 mM or resveratrol for 24 hours prior to overnight treatment with 100 μM 3-oxo-C12-HSL were examined by immunofluorescence microscopy staining for the tight junction protein, ZO-1. Treatment with 3-oxo-C12-HSL distorted intercellular distribution of ZO-1. These changes were attenuated in cells pretreated with resveratrol or metformin. 60x magnification, Scale bar = 50 μm. (E–J) Calu-3 were cells grown on transwell inserts and pre-treated overnight with either vehicle control, metformin 1 mM (D–F) or resveratrol 20 μM (G–I). Transepithelial electrical resistance (TEER) was then measured at baseline and at regular intervals following treatment with 100 μM 3-oxo-C12-HSL (E,H). 3-oxo-C12-HSL significantly reduced TEER within 2 hours and this effect persisted for at least 48 hours. Pre-treatment with metformin lead to restoration of barrier integrity at 24 (E) and 48 (F) hours despite 3-oxo-C12-HSL treatment. A similar effect was seen with resveratrol at 24 (H) and 48 (I) hours. (E–J) Primary NhBEs cultured on transwell inserts were pretreated with either vehicle control, metformin 1 mM (K-M), or resveratrol 20 μM (N–P). TEER was then measured before and at 3, 6, 12, and 24 hours following treatment with 100 μM 3-oxo-C12-HSL. Results are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test, n = 5 independent experiments (A–C) or two-way ANOVA with Tukey’s multiple comparisons test, n = 5 independent experiments (E–J) n = 6 (K–P).

Figure 9

Schematic of the effect of P. aeruginosa QS molecules on lung epithelial host response. (A) QS molecules disrupt mitochondrial bioenergetics, attenuate cellular respiration, induce ROS generation and the apoptosis pathway, repress the PGC-1α-TFAM mitochondrial biogenesis pathway, and trigger loss of barrier integrity. (B) Therapy targeting activation of the PGC-1α pathway via genetic or pharmacologic approaches, partially rescues the impairments in mitochondrial respiration, mitochondrial biogenesis, and barrier integrity.