Laboratory of Biofunctional Design Chemistry, Institute for Chemical Research, Kyoto University, Gokasho, Uji 611-0011, Japan.
Laboratory of Molecular Microbial Science, Institute for Chemical Research, Kyoto University, Gokasho, Uji 611-0011, Japan.
J Mol Biol. 2020 Nov 6;432(22):5876-5888. doi: 10.1016/j.jmb.2020.09.009. Epub 2020 Sep 12.
Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.
细胞外膜囊泡 (EMV) 是生物源性分泌脂质囊泡，在与人类疾病和细菌发病机制相关的细胞间通讯中发挥重要作用。它们正在被研究用于诊断、疫苗和生物技术。然而，现有的方法存在许多问题。高速离心是一种广泛用于收集 EMV 的方法，但可能会导致结构伪影。免疫染色方法需要多个步骤，因此从分泌细胞中分离和检测 EMV 非常耗时。此外，使用这些方法检测 EMV 需要特定且昂贵的抗体。为了解决这些问题，开发一种简单快速的检测方法，无需从分泌细胞中分离即可检测培养介质中的 EMV，是一项紧迫的任务。在这项研究中，我们以革兰氏阴性菌 Shewanella vesiculosa HM13 为模型，该菌产生大量的 EMV，包括一种具有高纯度的货物蛋白。曲率感应肽被用作 EMV 检测工具。FAAV 是一种源自分选连接蛋白 1 的肽，即使在复杂的培养介质中存在分泌细胞，它也能选择性地结合 EMV。FAAV 可以在几分钟内完全检测到 EMV，并且 FAAV 对蛋白酶的抗性使其能够在培养介质中长时间使用。荧光/荧光能量转移被用于开发一种高灵敏度检测 EMV 量变化的方法。总体而言，我们的结果表明 FAAV 有潜力用于原位检测培养介质中的 EMV。
