College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 34134, Republic of Korea.
College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 34134, Republic of Korea.
Fish Shellfish Immunol. 2024 May;148:109508. doi: 10.1016/j.fsi.2024.109508. Epub 2024 Mar 20.
Bacterial extracellular vesicles (BEVs) are nanosized structures that play a role in intercellular communication and transport of bioactive molecules. Streptococcus parauberis is a Gram-positive pathogenic bacterium that causes "Streptococcosis" in fish. In this study, we isolated S. parauberis-derived extracellular vesicles (SpEVs), and then physicochemical and immunomodulatory properties were determined to elucidate their biological functions. Initially, the biogenesis of SpEVs was detected using field emission scanning electron microscopy, which revealed that secretory phase SpEVs attached to the outer surface of S. parauberis. SpEVs had an average particle diameter and zeta potential of 168.3 ± 6.5 nm and -17.96 ± 2.11 mV, respectively. Field emission transmission electron microscopy analysis confirmed the presence of round or oval-shaped SpEVs with clear membrane margins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed three sharp protein bands when SpEVs were stained with Coomassie blue. In vitro toxicity of SpEVs was assayed using the murine macrophage RAW 264.7 cells and we observed no significant (p < 0.05) viability reduction up to 50 μg/mL qRT-PCR results revealed that SpEVs-treated (5 and 10 μg/mL) RAW 264.7 cells significantly (p < 0.05) induced the mRNA of proinflammatory (Il1β, Il6, and Tnfα) and anti-inflammatory (Il10) cytokines in a concentration-dependent manner. In vivo immunomodulatory effects of SpEVs were investigated by injecting SpEVs (5 and 10 μg/fish) into adult zebrafish. Transcriptional analysis based on qRT-PCR indicates significant (p < 0.05) upregulation of proinflammatory (il1β, il6, and tnfα) and anti-inflammatory (il10) genes in a concentration-dependent manner in zebrafish kidney. Further, protein expression results in zebrafish spleen tissue confirmed the immunomodulatory activity of SpEVs. In conclusion, SpEVs display the characteristics of BEVs and immunomodulatory activities, suggesting their potential application as vaccine candidate.
细菌细胞外囊泡（BEVs）是一种在细胞间通讯和生物活性分子运输中发挥作用的纳米结构。猪链球菌副猪亚种是一种革兰氏阳性致病性细菌，可导致鱼类“链球菌病”。在这项研究中，我们分离了猪链球菌副猪亚种来源的细胞外囊泡（SpEVs），然后测定了其物理化学和免疫调节特性，以阐明其生物学功能。最初，使用场发射扫描电子显微镜检测 SpEVs 的生物发生，结果显示分泌期 SpEVs 附着在猪链球菌副猪亚种的外表面。SpEVs 的平均粒径和 Zeta 电位分别为 168.3±6.5nm 和-17.96±2.11mV。场发射透射电子显微镜分析证实了具有清晰膜边缘的圆形或椭圆形 SpEVs 的存在。当 SpEVs 用考马斯亮蓝染色时，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示出三条尖锐的蛋白质带。使用鼠巨噬细胞 RAW 264.7 细胞测定 SpEVs 的体外毒性，我们观察到在高达 50μg/mL 的浓度下，细胞活力没有显著降低（p<0.05）。qRT-PCR 结果显示，SpEVs 处理（5 和 10μg/mL）的 RAW 264.7 细胞以浓度依赖的方式显著（p<0.05）诱导促炎（Il1β、Il6 和 Tnfα）和抗炎（Il10）细胞因子的 mRNA。通过向成年斑马鱼注射 SpEVs（5 和 10μg/鱼）来研究 SpEVs 的体内免疫调节作用。基于 qRT-PCR 的转录分析表明，在斑马鱼肾脏中，促炎（il1β、il6 和 tnfα）和抗炎（il10）基因的表达呈浓度依赖性显著上调（p<0.05）。此外，在斑马鱼脾脏组织中的蛋白质表达结果证实了 SpEVs 的免疫调节活性。总之，SpEVs 显示出 BEVs 的特征和免疫调节活性，表明它们有作为疫苗候选物的应用潜力。
