Endophthalmitis is a sight-threatening infection and a serious consequence of complications during intraocular surgery or penetrating injury of which Pseudomonas aeruginosa is an important etiology. Extracellular vesicles (EVs) have evolved as a promising entity for developing diagnostic and therapeutic biomarkers due to their involvement in intracellular communication and pathogenesis of diseases. We aimed to characterise the protein cargo of extracellular vesicles, isolated from a murine (C57BL/6) model of P. aeruginosa endophthalmitis by LC-MS/MS at 24 h post infection (p.i). EVs were extracted by ultracentrifugation, characterized by Dynamic Light Scattering (DLS) and western blotting with tetraspannin markers, CD9 and CD81 and quantified by the ExoCet quantification kit. Multiplex ELISA was performed to estimate the levels of TNF-α, IL-6, IFN-γ and IL-1β. Proteomic analysis identified 2010 proteins (FDR ≤0.01) in EVs from infected mice eyes, of which 137 were differentially expressed (P-value ≤ 0.05). A total of 101 proteins were upregulated and 36 were downregulated. Additionally, 43 proteins were exclusive to infection set. KEGG and Gene Ontology revealed, Focal adhesion, Phagosome pathway, Complement cascade and IL-17 signalling pathway are crucial upregulated pathways involving proteins such as Tenascin, caveolin 1, caveolin 2, glutamine synthetase, microtubule-associated protein, C1, C8 and IL-17. Tenascin and caveolins are known to suppress anti-inflammatory cytokines further exacerbating the disease. The result of this study provides insight into the global extracellular vesicle proteome of P. aeruginosa endophthalmitis with their functional correlation and distinctive pattern of expression and tenascin, caveolin 1 and caveolin 2 are attractive biomarkers for P. aeruginosa endophthalmitis.
Keywords: Endophthalmitis; Extracellular vesicles; Mass spectrometry; Proteomics; Pseudomonas aeruginosa.
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