Pseudomonas aeruginosainfection increases palmitoyl carnitine release by host-derived extracellular vesicles

Abstract

Pseudomonas aeruginosa(PA), an opportunistic gram-negative pathogen, is the most common pathogen identified in all culture positive cases of infectious keratitis. Extracellular vesicles (EVs) are released by most cells in the body and function in intercellular communication. We have previously reported a change in the proteome of host-derived EVs from corneal epithelial cells during PA infection. In the present study, we investigated changes in the metabolome of host-derived EVs from PA infected (PA-C EVs) and non-infected cells (C EVs). We found that one metabolite, palmitoyl carnitine (PAMC), was significantly upregulated in PA-C EVs. To determine the significance of PAMC release, we investigated the effect of PAMC treatment on corneal epithelial cells and neutrophils. EVs were isolated from culture media using size exclusion chromatography. EVs were then characterized using nanoparticle tracking analysis, transmission electron microscopy, and western blot. Metabolomics was performed using an untargeted approach. We found that palmitoyl carnitine (PAMC) was the most abundant metabolite present in PA-C EVs and was increased more than 3 fold compared to C EVs. Treatment of corneal epithelial cells with increasing levels of PAMC increased nuclear translocation of the NF-κB subunit p65. This was associated with an increase in IL-8 production and neutrophil migration. PAMC also increased levels of mitochondrial calcium. Upon inoculation of corneal epithelial cells with PA, 50 μM PAMC completely eradicated intracellular PA, but stimulated growth of extracellular PA. Taken together, these findings suggest that PA exploits EV release by host cells to deplete PAMC from the intracellular environment.