June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Characterization and proteome profiling of extracellular vesicles in a murine model of Pseudomonas aeruginosa endophthalmitis
Author Affiliations & Notes
  • Dhanwini Rudraprasad
    Jhaveri Microbiology Centre, LV Prasad Eye Institute, Hyderabad, Telangana, India
    Manipal Academy of Higher Education, Manipal, Karnataka, India
  • Aravind Kumar Rengan
    Indian Institute of Technology Hyderabad, Hyderabad, Telangana, India
  • Milind Neelkanth Naik
    Ophthalmic Plastic Surgery & Facial Aesthetics, LV Prasad Eye Institute, Hyderabad, Telangana, India
  • Joveeta Joseph Ruben
    Jhaveri Microbiology Centre, LV Prasad Eye Institute, Hyderabad, Telangana, India
  • Footnotes
    Commercial Relationships   Dhanwini Rudraprasad None; Aravind Rengan None; Milind Naik None; Joveeta Joseph Ruben None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 858. doi:
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      Dhanwini Rudraprasad, Aravind Kumar Rengan, Milind Neelkanth Naik, Joveeta Joseph Ruben; Characterization and proteome profiling of extracellular vesicles in a murine model of Pseudomonas aeruginosa endophthalmitis. Invest. Ophthalmol. Vis. Sci. 2022;63(7):858.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Extracellular vesicles (EVs) are representators of the cellular microenvironment and mediate intercellular communication and pathogenesis. Here, we investigate the protein topology of EVs derived from C57BL/6 murine model of P. aeruginosa endophthalmitis with an aim to understand the pathobiology of the disease.

Methods : Endophthalmitis was induced by intravitreal injection of Pseudomonas aeruginosa in C57BL/6 mice followed by enucleation at 24 hours. EVs were extracted by ultracentrifugation, characterized by DLS and western blotting with tetraspannin markers, CD9 and CD81, and quantified by ExoCet quantification kit. EVs were trypsin digested and subjected to LC-MS/MS. The proteins were analysed for its biological process and protein class by Gene Ontology (GO) and identification of enriched pathways by Kyoto Encyclopedia of Genes and Genomes (KEGG). Statistical analysis was performed by Unpaired t-test and all values represent mean ± SD for 3 biological replicates per time point.

Results : DLS and western blot confirmed the presence of EVs having a mean diameter of 332.2±93nm in infected eyes, while the EVs from the control set was 362±40 nm. Total number of EVs in infected eyes was 1.5X1010 (±2.6X109), compared to 6.8X109 (±3.6X109) in control eyes. Proteome analysis of these EVs identified 2010 proteins (FDR ≤0.01) in infected eyes, of which 131 were differentially expressed (DEPs) (P-value ≤0.05). A total of 101 proteins were upregulated and 36 were downregulated while 55 proteins were found to be unique to the infected set. KEGG and GO analysis revealed presence of Cav1, Cav2, Coronin 1A, Integrin β, Protein kinase C, Tubulin-α, Tubulin-β, Complement C8, G-protein signalling 9 and IL-17 in EV from infected eyes. While Cav1 and Cav2 are reported to regulate inflammatory response of the host, Calnexin, prolactin induced protein and thioredoxin-related transmembrane protein-1 were present significantly in the infection set, suggesting a major role in host immunity and protection against microbial infections.

Conclusions : This study is the first attempt to comprehensively investigate the global proteome of EVs derived from mice model of P. aeruginosa endophthalmitis. The EV proteins Cav1, Cav2, Coronin 1A, Integrin β, type I keratin are known mediators of pathogenesis and are most suitable to study them as potential prognostic markers for endophthalmitis.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.


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