Abstract
Purpose :
Pseudomonas aeruginosa (PA) is a major cause of infectious keratitis. Extracellular vesicles (EVs) are membrane bound nanovesicles secreted by cells that play a key role in cell communication. The purpose of this study is to characterize the biomolecular cargo in corneal epithelial-derived EVs released during PA infection in the corneal epithelium.
Methods :
Telomerase-immortalized human corneal epithelial cells were infected with a standard invasive test strain of PA (strain PA01) for six hours. EVs were isolated from cell culture supernatants by size exclusion chromatography. Pooled fractions F1/F2 contained EVs and pooled fractions F4/F5 contained secretory proteins. Uninfected cells and planktonic PA were used as controls. Immunoblotting was performed for EV markers CD63, CD9, CD81, TSG101 and flotillin; size and concentration were determined by nanoparticle tracking analysis (NTA). Mass spectrometry was performed by the Proteomics Core at UTSW. Protein abundance values were log2 transformed and analyzed using a two-tailed t-test. Proteins with p<0.05 were subject to analysis using functional enrichment analysis tool (FunRich) software.
Results :
All of the tested EV markers were detected in EVs. Protein concentration was lower in EVs than the secretory fraction. Using NTA, the average EV particle size was 30 ± 150 nm. PA infection did not alter EV particle number. Comapring protein abundance, 132 proteins in EVs released from PA-infected cells were significantly different than EV released from non-infected cells. Of these, 72 proteins were upregulated in response to PA-infection. Gene enrichment analysis indicated that among the 132 differentially expressed proteins, most genes belong to energy and metabolic pathways. Of the 72 upregulated proteins, gene enrichment analysis showed that the highest percentage of genes were related to G alpha and IL-8 signaling, energy pathways, signal transduction, metabolism, and hydrolase activity.
Conclusions :
While PA infection does not alter the number of EVs released by corneal epithelial cells, it does alter the proteomic profile. Bioinformatics analysis indicates that proteins related to critical biological process including immune signaling, energy metabolism and cell communication pathways are enriched in corneal epithelial EVs released during PA infection.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.