Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, Maharashtra, India.
Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan.
Methods Mol Biol. 2024;2843:119-136. doi: 10.1007/978-1-0716-4055-5_8.
Bacterial extracellular vesicles (BEVs) are nano- or micrometer-sized membrane-bound lipid vesicles released from both Gram-negative and Gram-positive bacteria. Cellular transport, communication, pathogenesis, and host-pathogen interactions are some of the major biological processes impacted by BEVs. Among these, host-pathogen interactions and bacterial pathogenesis are emerging as highly important targetable avenues underlined by the issues of antimicrobial resistance, thus demanding novel targets and approaches to treat bacterial infections. In this aspect, the study of the interaction of BEVs with bacteria and/or host cells becomes imperative and brings the membrane fusion process to the forefront. Furthermore, membrane fusion also underscores the performance of BEVs as nano-therapeutic delivery platforms. Here, we report methods to study fusion kinetics between mycobacteria-derived extracellular vesicles, which we refer to as MEVs, and intact mycobacteria or MEVs themselves. We also discuss the isolation of MEVs and their characterization. We outline critical factors that affect fusion kinetics by MEVs. The same principle can be extended for studying fusion between BEVs and mammalian host cells important for understanding how BEVs influence host-pathogen crosstalk.
细菌细胞外囊泡 (BEV) 是由革兰氏阴性菌和革兰氏阳性菌释放的纳米或微米大小的膜结合脂质囊泡。细胞运输、通讯、发病机制和宿主-病原体相互作用是受 BEV 影响的一些主要生物学过程。在这些过程中，宿主-病原体相互作用和细菌发病机制作为具有高度靶向性的途径出现，这是由抗微生物药物耐药性问题所强调的，因此需要新的靶点和方法来治疗细菌感染。在这方面，研究 BEV 与细菌和/或宿主细胞的相互作用变得至关重要，并将膜融合过程推到了前沿。此外，膜融合也突出了 BEV 作为纳米治疗递送平台的性能。在这里，我们报告了研究分枝杆菌来源的细胞外囊泡（我们称之为 MEV）与完整分枝杆菌或 MEV 自身之间融合动力学的方法。我们还讨论了 MEV 的分离及其特性。我们概述了影响 MEV 融合动力学的关键因素。同样的原理可以扩展到研究 BEV 与对理解 BEV 如何影响宿主-病原体串扰很重要的哺乳动物宿主细胞之间的融合。
