Abstract
Purpose :
Inter-organelle communication plays a crucial role in cellular signaling during pathogen infection, but has not yet been explored in corneal disease. Two key proteins involved in inter-organelle tethering include the mitofusins, mitofusion 1 (MFN1) and mitofusin 2 (MFN2). We have previously shown that PA infection induces robust mitochondrial fission in corneal epithelial cells, despite significant increases in MFN1 and MFN2. The objective of this study is to investigate the potential regulatory role of MFN1 and MFN2 in mitochondrial-endoplasmic reticulum (ER) tethering during PA keratitis and the subsequent effect on intracellular PA survival.
Methods :
A standard invasive strain of PA (PA01) was used for the study. Human telomerase immortalized corneal epithelial (hTCEpi) cells were infected with PA01 for 2 hrs. Western blot and immunofluorescence staining were used to measure changes in MFN1 and MFN2 expression during PA infection. The role of MFN1 and MFN2 on intracellular PA survival was assessed using siRNA followed by a gentamicin survival assay. Inter-organelle interactions between mitochondria and the ER were further visualized by immunostaining for HSP60 and calnexin using laser confocal microscopy. Mitochondrial DNA was quantified using droplet digital PCR.
Results :
PA infection induced the expression of mito-ER tethering proteins MFN1 and MFN2. This was associated with an increase in co-localization of the mitochondrial and ER proteins, HSP60 and calnexin. PA infection also decreased mitochondrial mass. siRNA knockdown of MFN1 and MFN2 reduced mito-ER contact sites, attenuated the PA-mediated decrease in mitochondrial mass and reduced intracellular levels of PA.
Conclusions :
Taken together, mito-ER tethering appears to increase during the early stages of PA invasion. Disruption of inter-organelle contact sites through the selective reduction of MFNs is beneficial to host cells. Further studies are needed to elucidate the mechanism(s) that underly the formation of mito-ER tethers and their role in host-pathogen interactions in the cornea.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.