外膜囊泡(OMV)是革兰氏阴性细菌脱落的挤出纳米结构,含有周质内容物,通常包括具有免疫原性的毒力因子。为了评估它们用于疫苗开发的潜力,我们从坏死梭菌亚种坏死中纯化了OMV,机会性坏死感染病原体,并使用蛋白质组学表征了这些结构,脂质谱分析,和细胞毒性测定。对密度梯度纯化的F.necrophorumOMVs的蛋白质组学分析鉴定出342种蛋白质,其中很大一部分是外膜蛋白(OMPs),其次是细胞质蛋白,基于亚细胞定位预测分析。OMP和毒素是鉴定出的强度最高的蛋白质之一,包括43kDa-OMP-,OmpA-,和OmpH家族蛋白,细胞表面蛋白,FadA粘附蛋白,白细胞毒素-LktA-家族丝状粘附素,血凝素的N端结构域,以及OMP转运蛋白和组装因子。Western印迹分析证实了坏死F.OMV中存在几种OMP和毒素。脂质谱分析显示磷脂,鞘脂,和乙酰肉碱作为OMV的主要脂质内容物。乳酸-脱氢酶-细胞毒性测定显示OMV对牛B淋巴细胞系(BL-3细胞)具有高度的细胞毒性。因此,我们的数据提示需要进一步的研究来评估OMV诱导免疫应答的能力,并评估其体内疫苗的潜力.
Outer-membrane vesicles (OMVs) are extruded nanostructures shed by Gram-negative bacteria, containing periplasmic contents, and often including virulence factors with immunogenic properties. To assess their potential for use in vaccine development, we purified OMVs from the Fusobacterium necrophorum subspecies necrophorum, an opportunistic necrotic infection-causing pathogen, and characterized these structures using proteomics, lipid-profiling analyses, and cytotoxicity assays. A proteomic analysis of density-gradient-purified F. necrophorum OMVs identified 342 proteins, a large proportion of which were outer-membrane proteins (OMPs), followed by cytoplasmic proteins, based on a subcellular-localization-prediction analysis. The OMPs and toxins were among the proteins with the highest intensity identified, including the 43-kDa-OMP-, OmpA-, and OmpH-family proteins, the cell-surface protein, the FadA adhesin protein, the leukotoxin-LktA-family filamentous adhesin, the N-terminal domain of hemagglutinin, and the OMP transport protein and assembly factor. A Western blot analysis confirmed the presence of several OMPs and toxins in the F. necrophorum OMVs. The lipid-profiling analysis revealed phospholipids, sphingolipids, and acetylcarnitine as the main lipid contents of OMVs. The lactate-dehydrogenase-cytotoxicity assays showed that the OMVs had a high degree of cytotoxicity against a bovine B-lymphocyte cell line (BL-3 cells). Thus, our data suggest the need for further studies to evaluate the ability of OMVs to induce immune responses and assess their vaccine potential in vivo.