Effect of ULK1 Inhibition on Corneal Epithelial Cells During Pseudomonas aeruginosa Infection

INTRODUCTION: Pseudomonas aeruginosa (PA) keratitis is a severe infection of the cornea that can lead to blindness. Studies in our lab have shown that PA exploits autophagy, a major cellular degradation process, in corneal epithelial cells (hTCEpi cells) to promote intracellular survival. We have further shown that the inhibition of autophagy by the Unc 51-like kinase (Ulk1), an enzyme that mediates formation of the autophagosome, reduces intracellular levels of PA. More recently, we have demonstrated that PA infection negatively impacts host mitochondria. ULK1/2 has been reported to translocate to mitochondria to mediate mitophagy however, a role for ULK1/2 in mitochondrial homeostasis during infection has not yet been explored. In this study, we investigated the effects of the inhibition of Ulk1 during PA infection on host mitochondria. METHODS: Telomerase-immortalized human corneal epithelial (hTCEpi) cells were used for this study. Cells were cultured in serum-free defined keratinocyte media with growth supplements. Cells were inoculated with 106 CFU/ml of PA in log growth phase with or without treatment with 1 �M of the Ulk1/2 inhibitor MRT68921. Intracellular levels of PA were quantified using a gentamicin survival assay. Oxygen consumption and mitochondrial polarization were assessed using Seahorse metabolic flux analysis and tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1), respectively. Levels of pro-inflammatory cytokines were assessed using ELISA. Untargeted metabolomics was performed using mass spectrometry. Cellular changes were further evaluated using transmission electron microscopy (TEM). RESULTS: PA infection induced robust mitochondrial depolarization. There was a corresponding increase in secretion of IL-6 and IL-8. Treatment with MRT68921 restored mitochondrial polarization and reduced IL-6, but had no effect on IL-8. MRT68921 also reduced intracellular levels of PA. TEM demonstrated robust mitochondrial fission during PA infection. Treatment with MRT68921 preserved mitochondrial structure and polarization during PA infection. CONCLUSIONS: Taken together, these data suggest that Ulk1/2 modulates the host mitochondrial response to PA infection. Further studies are needed to determine the mechanism by which MRT68921 preserves mitochondrial function and its potential use as an adjunct therapeutic for PA-mediated keratitis.