Biology subjects

Ayilam Ramachandran, R.

Publications and source records attributed to Ayilam Ramachandran, R..

2 recordsLinked to original sources

bioRxiv2024

Pseudomonas aeruginosa infection increases palmitoyl carnitine release by host-derived extracellular vesicles

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-{kappa}B 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 M 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.

microbiology↗

bioRxiv2024

Pseudomonas aeruginosa impairs mitochondrial function and metabolism during infection of corneal epithelial cells

Pseudomonas aeruginosa (PA) is a gram-negative opportunistic pathogen that can infect the cornea as a result of trauma or contact lens wear. In addition to their known energy producing role, mitochondria are important mediators of immune signaling and host defense. While certain pathogens have developed strategies to evade host defenses by modulating host mitochondrial dynamics and metabolism, the ability of PA to harness host cell mitochondria during corneal infection is unknown. Using a combination of biochemical and imaging techniques, we show that PA infection of corneal epithelial cells induced mitochondrial fission in a DRP1-dependent manner that preceded PINK1/Parkin and FUNDC1-mediated mitophagy. PA also impaired NADH-linked respiration through a reduction in complex 1. This corresponded to a decrease in metabolic pathways related to glycolysis and the TCA cycle. Metabolomics analysis further demonstrated an upregulation of the pentose phosphate pathway, arginine, purine, and pyrimidine metabolism in PA infected cells. These pathways may provide a key source of nucleotides, amino acids, and nitrogen for both the host cell and PA, in addition to antioxidant functions. Following treatment with gentamicin to kill all extracellular bacteria, metabolic flux analysis showed that corneal epithelial cells were able to restore mitochondrial function despite the continued presence of intracellular PA. Taken together, these data demonstrate that mitochondrial dysfunction and metabolic rewiring in host cells is triggered by extracellular PA, but once inside, PA requires healthy mitochondria to ensure host cell survival.

microbiology↗