Abstract
Purpose :
Pseudomonas aeruginosa (PA) is an opportunistic gram negative pathogen that can cause severe infectious keratitis. Mitochondria are important regulators of energy homeostasis and host-pathogen interactions during infection. This study investigated the effects of PA infection on mitochondrial and metabolic homeostasis in the corneal epithelium.
Methods :
A standard invasive test strain of PA (strain PA01) was used to infect telomerase-immortalized human corneal epithelial (hTCEpi) cells for 2 hours. Moxifloxacin was used to kill both intra- and extracellular bacteria, gentamicin treatment killed all extracellular bacteria, and methyl-β-cyclodextrin (MβCD) treatment attenuated PA01 internalization into hTCEpi cells. Mitochondrial metabolism was quantified using a seahorse metabolic flux analyzer. Mtochondrial polarization and morphological changes were characterized using JC1 staining and transmission electron microscopy (TEM), respectively. Mitophagy pathways were evaluated by western blot.
Results :
PA01 infection of hTCEpi cells altered mitochondrial morphology and triggered mitochondrial depolarization. These effects were blocked by treatment with either antibiotic or MβCD. Metabolic flux analysis showed an increase in both the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) in PA01 infected cells. Maximal respiration was decreased. OCR and ECAR were unchanged compared to controls in PA01-infected cells treated with moxifloxacin. Treatment with MβCD, which reduces intracellular levels of PA, partially abrogated the effect of PA01 infection on metabolism. Changes were also observed in the mitophagy pathway.
Conclusions :
PA invasion alters metabolic homeostasis by inducing robust mitochondrial depolarization and a corresponding reduction in oxidative phosphorylation in hTCEpi cells. These changes are not mediated by the presence of intracellular bacteria but are associated with changes in the plasma membrane that occur during the internalization process. Together, these findings suggest that cell membrane-mitochondrial cross talk may regulate important host responses during PA infection in the corneal epithelium.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.