Jhaveri Microbiology Centre, Brien Holden Eye Research Centre, L. V. Prasad Eye Institute, Hyderabad, Telangana, India; Manipal Academy of Higher Education, Manipal, Karnataka, India.
Ophthalmic Plastic Surgery & Facial Aesthetics, LV. Prasad Eye Institute, Hyderabad, Telangana, India.
Exp Cell Res. 2022 Oct 1;419(1):113306. doi: 10.1016/j.yexcr.2022.113306. Epub 2022 Aug 10.
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.
眼内炎是一种威胁视力的感染，也是眼内手术或穿透性损伤并发症的严重后果，其中铜绿假单胞菌是重要的病因。由于细胞内通讯和疾病发病机制中的作用，细胞外囊泡 (EVs) 已成为开发诊断和治疗生物标志物的有前途的实体。我们旨在通过 LC-MS/MS 对感染后 24 小时 (p.i.) 的铜绿假单胞菌眼内炎的 C57BL/6 小鼠模型分离的细胞外囊泡的蛋白质货物进行特征分析。通过超速离心提取 EVs，用四跨膜蛋白标志物、CD9 和 CD81 的 Western 印迹和 ExoCet 定量试剂盒进行表征，并通过多重 ELISA 估计 TNF-α、IL-6、IFN-γ 和 IL-1β 的水平。蛋白质组学分析鉴定出感染小鼠眼睛 EVs 中的 2010 种蛋白质 (FDR ≤0.01)，其中 137 种差异表达 (P 值≤0.05)。共有 101 种蛋白上调，36 种蛋白下调。此外，43 种蛋白是感染组特有的。KEGG 和基因本体论揭示，焦点粘附、吞噬体途径、补体级联和 IL-17 信号通路是关键的上调途径，涉及 Tenascin、窖蛋白 1、窖蛋白 2、谷氨酰胺合成酶、微管相关蛋白、C1、C8 和 IL-17 等蛋白质。Tenascin 和窖蛋白已知可抑制抗炎细胞因子，从而进一步加重疾病。这项研究的结果提供了铜绿假单胞菌眼内炎的细胞外囊泡蛋白质组的全局视图，及其功能相关性和独特的表达模式，Tenascin、窖蛋白 1 和窖蛋白 2 是铜绿假单胞菌眼内炎有吸引力的生物标志物。
