Abstract
Purpose :
Endophthalmitis occurs from the entry of pathogens after trauma or intraocular surgery and prompt diagnosis is vital for improved visual outcome. Lately, Extracellular Vesicles (EVs) have been applauded for its potential as disease biomarkers. The purpose of this study was to understand the protein cargo of EVs in murine model of bacterial and fungal endophthalmitis.
Methods :
EVs were isolated by differential ultracentrifugation from C57BL/6 mice eyes infected with bacterial(P. aeruginosa–PA; S. aureus-SA) and fungal(A. flavus–AF; C. albicans-CA) pathogens. Isolated EVs were characterized and Mass Spectrometry(LC-MS/MS) analysis was performed for comparative proteome profile of EVs derived from bacterial versus fungal infected mice.
Results :
The average size of the EVs from bacteria-infected mice was 317±56nm, while the average diameter of EVs from fungal-infected mice was 203±30nm. The quantity of EVs was significantly higher in the infected group compared to the control group (p<0.05). Compared to EVs derived from control mice, EVs from infected group were enriched for EV tetraspanin markers CD9, CD63, and CD81. Further, the proteomic analysis identified proteins significantly expressed in both bacterial group PA-170; SA-40 and fungal group AF-113; CA-78. Comparative (bacteria versus fungal) study revealed that the protein cargo of the EVs was very distinct among the infected sets. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that in bacterial infections, proteins such as synapsin-1, complement cascade C8-α, calpain-2, and microtubule associated proteins 1A/1B are involved in pathogenesis of endophthalmitis and KEGG analysis showed that these proteins were associated with pathways such as MAPK, phagosome, and focal adhesion. Similarly, proteins such as Proteasome activator-2, Interleukin enhancer-binding factor-2 and Gasdermin-A were exclusively present in fungal infections, and pathways associated were microbial, and carbon metabolism. However, proteins such as Kinase-C-β, Phospholipase-B, and sorting nexin-29 were common to both fungal and bacterial infections.
Conclusions :
Our findings collectively demonstrate that EV proteins can delineate information on disease pathobiology. Further validation of these proteins can serve as important signature molecules for diagnosing bacterial and fungal endophthalmitis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.