College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 34134, Republic of Korea.
Jeju Bio Research Center, Korea Institute of Ocean Science and Technology, Gujwa-eup, Jeju 2670, Republic of Korea.
Fish Shellfish Immunol. 2024 Nov;154:109945. doi: 10.1016/j.fsi.2024.109945. Epub 2024 Oct 6.
Bacterial extracellular vesicles (BEVs) are nanoscale membrane-bound structures involved in intercellular communication and transport of bioactive molecules. In this study, we described the proteomic insight and anti-inflammatory activity of Streptococcus parauberis BEVs (SpEVs). Proteomics analysis of SpEVs identified 6209 distinct peptides and 1039 proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated enrichment in pathways related to the biosynthesis of aminoacyl tRNA, amino acids, and secondary metabolites. Based on the predicted protein-protein interactions, we discovered key immunological proteins such as IL12A, IL12B, IL8, CD28, and NF-κB between SpEVs and human proteins. Functionally, SpEVs exhibit strong anti-inflammatory activity in LPS-stimulated Raw 264.7 cells by reducing the production of key inflammatory mediators. These include nitric oxide (NO), reactive oxygen species (ROS), inflammatory cytokines such as TNFα and IL6, as well as inflammation-related proteins like inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). qRT-PCR and immunoblotting results clearly indicate that SpEVs modulate the NF-κB and MAPK pathways to induce anti-inflammatory activity. Furthermore, in vivo experiments with zebrafish larvae demonstrated that SpEVs treatment reduced the NO and ROS production with minimal cell mortality. Finally, we validated the anti-inflammatory activity of SpEVs in vivo by systematically assessing the inhibition of NO production, reduction in ROS generation, prevention of cell death, and modulation of NF-κB and MAPK signaling pathways. In conclusion, SpEVs contain rich in unique proteins that play crucial roles in mediating anti-inflammatory effects.
细菌细胞外囊泡（BEVs）是参与细胞间通讯和生物活性分子运输的纳米级膜结合结构。在这项研究中，我们描述了副猪链球菌 BEV（SpEVs）的蛋白质组学见解和抗炎活性。SpEVs 的蛋白质组学分析鉴定出 6209 个独特肽段和 1039 种蛋白质。京都基因与基因组百科全书（KEGG）分析表明，这些蛋白质与氨酰 tRNA、氨基酸和次生代谢物的生物合成途径相关。基于预测的蛋白质-蛋白质相互作用，我们发现了 SpEVs 与人类蛋白质之间的关键免疫蛋白，如 IL12A、IL12B、IL8、CD28 和 NF-κB。功能上，SpEVs 在 LPS 刺激的 Raw 264.7 细胞中通过减少关键炎症介质的产生表现出强烈的抗炎活性。这些介质包括一氧化氮（NO）、活性氧（ROS）、肿瘤坏死因子-α（TNFα）和白细胞介素-6（IL6）等炎症细胞因子，以及诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）等炎症相关蛋白。qRT-PCR 和免疫印迹结果清楚地表明，SpEVs 通过调节 NF-κB 和 MAPK 通路诱导抗炎活性。此外，斑马鱼幼虫的体内实验表明，SpEVs 处理可减少 NO 和 ROS 的产生，同时细胞死亡率最低。最后，我们通过系统评估 NO 产生的抑制、ROS 生成的减少、细胞死亡的预防以及 NF-κB 和 MAPK 信号通路的调节，在体内验证了 SpEVs 的抗炎活性。总之，SpEVs 含有丰富的独特蛋白质，这些蛋白质在介导抗炎作用中发挥着关键作用。
