Abstract
Purpose :
Pseudomonas aeruginosa (PA) is one of the most common pathogens associated with infectious keratitis, a devastating corneal condition that can lead to blindness. AMP-activated protein kinase (AMPK) is a central enzyme that is activated in low energy states, resulting in the promotion of many catabolic processes. AMPK activity has been shown to have diverse roles in the regulation of host defenses during pathogen invasion, but the effects are cell and pathogen specific. In this study, we investigated the role of AMPK in the modulation of mitochondrial function and bacterial survival in PA-infected corneal epithelial cells.
Methods :
Telomerase-immortalized human corneal epithelial (hTCEpi) cells were infected with the standard reference P. aeruginosa strain PA01 with or without co-treatment with increasing concentrations of the AMPK inhibitor BAY-3827 (0.1 μM, 1 μM, 10 μM). Intracellular levels of PA were quantified using a gentamicin survival assay. BAY-3827 cytotoxicity on hTCEpi cells was quantified using a lactate dehydrogenase (LDH) assay. Cell culture supernatants were assessed for IL-6 and IL-8 using an enzyme-linked immunoassay (ELISA). Mitochondrial polarization was measured by labeling with the cationic carbocyanine probe, JC-1, and imaged using laser confocal microscopy. Western blotting was used to evaluate NF-κB activation. Metabolomics was performed using liquid chromatography-mass spectroscopy.
Results :
The highest concentration of BAY-3827 (10 μM) resulted in decreased intracellular PA survival. This concentration of BAY-3827 had no cytotoxic effects on hTCEpi cells or PA alone. BAY-3827 significantly decreased levels of inflammatory markers, IL-6 and IL-8, in corneal epithelial cells infected with PA. PA infection resulted in a loss of mitochondrial membrane polarization and changes in mitochondrial morphology. Treatment with BAY-3827 abrogated these effects.
Conclusions :
Taken together, the inhibition of AMPK decreases bacterial invasion and maintains mitochondrial homeostasis during the early stages of PA infection. Further studies are needed to determine the mechanism by which AMPK promotes host cell defenses to PA.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.