Proteomic profiling of aspergillus flavus endophthalmitis derived extracellular vesicles in an in-vivo murine model

Abstract

Extracellular Vesicles (EVs) play pivotal roles in cell-to-cell communication, and are involved in potential pathological and physiological cellular processes. The aim of this study was to understand the proteomic cargo of these vesicles, in a murine model of Aspergillus flavus (AF) endophthalmitis. EVs were isolated from A. flavus infected C57BL/6 mice eyes by differential ultracentrifugation at 24 h post infection (p.i) and isolated EVs were characterized by Dynamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), Exocet assay, and western blot. Proteomic profiling of EVs was then evaluated by mass spectrometry (LC-MS/MS) and compared it with control uninfected mice. The average size of the EVs were 180-280 nm by DLS and the number of EVs increased to 1.55 × 1010 in infected mice in comparison to EVs from uninfected eye (1.24 × 109). Western blot was positive for CD9, CD63, and CD81 confirming the presence of EVs. LC-MS/MS analysis, identified 81 differentially expressed proteins, of these 22 were up-regulated and 59 were down-regulated. Gene Ontology (GO) analysis revealed enrichment of lipid metabolism, protein complex binding, and transferase activity, and the proteins associated were Aquaporin-5, CD177 antigen, Solute carrier family-25, and Calcium/calmodulin-dependent protein kinase. Additionally, KEGG pathway analysis indicated that glucagon signalling, metabolic, and PPAR signalling pathway were significantly associated with EVs from A. flavus infected mice eyes. The protein cargo in EVs from A. flavus endophthalmitis provides new insights into the pathogenesis of fungal endophthalmitis and validation of these proteins can serve as diagnostic and/or prognostic biomarkers for patients with a clinical suspicion of fungal endophthalmitis.

Lay summary: EVs play an important role in cell communication. In our study proteomic profiling of EVs isolated from A. flavus infected mice provided new insights into the understanding of the pathobiology of A. flavus endophthalmitis and validation of these proteins can serve as biomarkers.

Keywords: aspergillus flavus; extracellular vesicles; fungal endophthalmitis; inflammation; proteome.